Microplastics are widely present in the atmosphere, soil, and water and can be transferred, enriched, and bioaccumulated through the food chain. The choice of digestion solution depends on test sample characteristics and the digestion time varies from a few hours to several days, which may have detrimental effects on the original plastic by causing dissolution, fracture, and degradation, thereby potentially interfering with experimental data. Each existing detection technology has its advantages and limitations. To establish an efficient pre-separation treatment scheme for shellfish microplastics based on fluorescence staining technology, this study focused on the Manila clam (Ruditapes philippinarum) because they are one of the main indicators of microplastic pollution in shellfish in China. We investigated digestion efficiency, membrane obstruction rate, microplastic recovery rate, as well as changes in the infrared spectrum under two digestion systems: 10% potassium hydroxide (KOH) and protease K. Digestion conditions were determined and optimized accordingly. Furthermore, we combined the optimization scheme with Nile red fluorescence staining to validate the unknown fluorescent particles using a confocal micro-Raman spectrometer to confirm the applicability of the proposed scheme. The results showed that under a 10% KOH digestion system, there was an extremely significant difference in digestion efficiency among the three groups (P˂0.01). The highest digestion efficiency was observed in the 4-h group at (99.46±0.49)% with a blocking rate of (126.17±53.30)%. For the protease K digestion system, a significant difference was observed among the three groups (P˂0.05), with the average digestion efficiency being highest in the 16-h group at (99.66±0.08)%. No statistically significant differences were observed in recovery rates of standard microplastic particles among the experimental groups (P＞0.05). The major characteristic peaks observed between the experimental and quality control groups were consistent; however, differences existed in peak intensity, position, spectral quality, and other aspects. Based on the aforementioned comparative results, it is evident that under all experimental conditions, the most effective digestion occurred with 10% KOH at 50 ℃ for 4 h and protease K at 50 ℃ for 16 h. To enhance vacuum filtration time, we optimized the two digestion schemes by incorporating a pH adjustment using a 5% dilute hydrochloric acid solution after the initial treatment with 10% KOH at 50 ℃ for 4 h. Additionally, sodium laurylsulfonate (SDS) was added followed by ultrasound treatment for an additional duration of 30 min after digestion with protease K for 16 h. The experimental data demonstrated that the optimized KOH digestion scheme improved digestion efficiency to (99.80±0.06)% while reducing the membrane blockage rate to (95.78±6.51)%. The recovery rate of plastic standard samples with particle sizes ranging from 3 to 5 mm and 400 to 600 μm was (97.92±3.61)% and (89.58±4.14)%, respectively. For the optimized protease K digestion scheme, digestion efficiency improved and was found to be (99.97±0.02)% while the membrane blockage rate decreased to (94.37±9.85)%. The recovery rates for both plastic standards (PP, PS, PET and LDPE) were over 95% and 85%, respectively. The infrared spectrum changes observed in plastics following each treatment scheme were not significant. Therefore, the optimized method presented here is suitable for microplastic extraction in samples. After staining with Nile red solution, SDS ions possess adsorption activity on the surface of Nile red molecules, resulting in solubilization and formation of original micelles. This phenomenon leads to numerous false positive fluorescent bright spots within the optimized protease K group which may interfere with the micro-Raman measurement. However, the KOH optimization group did not exhibit the same occurrence, and microplastics detected in the sample were 0.99 g (wet weight). These included fibrous polyethylene terephthalate with a length of (971.23±22.01) μm and particle-like acrylonitrile butadiene styrene with a particle size of (26.88±1.69) μm. In conclusion, a 10% KOH solution was utilized as the digestion agent, followed by ultrasound treatment for 10 min. Subsequently, the solution was dissolved in a water bath at 50 ℃ and agitated at 150 rpm for 4 h. The pH was adjusted using a 5% dilute hydrochloric acid solution to achieve an optimal pre-treatment scheme. This approach was combined with Nile red fluorescence staining observation and confocal micro-Raman characterization, providing a simple, cost-effective, and efficient method for detecting and analyzing bivalve shellfish microplastics.