A high-performance liquid chromatography (HPLC) method was optimized for the determination of the free amino acids and flavor-enhancer nucleotides in aquatic products. The flavor-enhancer nucleotides and amino acids of ) were determined to provide basic data for exploiting shellfish seasoning compounds. After sample preparation using purified water, free amino acidsDabs-Cl) pre-column derivatization and reversed-phase HPLC. The best separation was obtained using a 17 mmol/L citric acid solution (pH 6.4, containing 4% dimethylformamide (DMF)) and acetonitrile (containing 4% DMF), gradient elution and a detection wavelength of 436 nm. Flavor (pH 4.5, 5 mmol/L tetramethylammonium hydroxide (TMAOH)) and acetonitrile, and a detection wavelength of 254 nm. This method displayed better precision and higher accuracy in meeting the analytical requirements. Recoveries varied from 90.2% to 108% and RSD varied from 0.7% to 7.1%. The results showed that high levels of taurine, and glutamic acid were found in Pacific oyster; glycine, taurine and alanine were the principal free amino acids in Farrer’s scallop and high percentages of glycine, taurine and alanine were detected in sunray surf clam. The taste activity values (TAVs) of alanine and glutamic acid in the three shellfish were >1; only the TAVs of glycine in Farrer’s scallop and sunray surf clam were >1, indicating that they make a significant contribution to taste. Regarding flavor-enhancing nucleotides, Pacific oyster contained more (IMP), accounting for 80%, while Farrer’s scallop contained the highest adenosine monophosphate (AMP) level followed by IMP; sunray surf clam contained IMP only. Although the concentration of flavor-enhancing nucleotides in three shellfish were low and TAVs were <1, the synergistic effect between nucleotides and amino acids has an important impact on shellfish flavor. Pacific oyster, Farrer’s scallop and sunray surf clam from offshore Yantai are rich in taste, and the flavor-enhancing nucleotides and amino acids, such as taurine, are valuable for human health. The study also provides basic data for the extraction of active substances for the preparation of seafood seasoning compound