White spot syndrome virus (WSSV) poses a significant threat to red swamp crayfish (Procambarus clarkii) aquaculture, a major crustacean industry in China. This virus' ability to remain latent in hosts and its complex transmission pathways make prevention and early detection crucial. However, current diagnostic methods, including the WOAH-recommended nested PCR, have limitations in speed, sensitivity, and convenience. This study aimed to develop a rapid, sensitive, and specific one-step conventional PCR assay for WSSV detection in P. clarkii, addressing the need for improved surveillance and early intervention strategies. In our study, specific primers (V1) were designed to target a 196 bp region within the WSSV vp19 gene. The assay's analytical specificity was rigorously evaluated using DNA/cDNA from 10 different pathogens in crayfish (5 viruses and 5 bacteria). To determine analytical sensitivity, serial dilutions of a recombinant plasmid (pMD19T-V1) containing the vp19 target sequence were employed. WOAH recommended TaqMan qPCR was used as the reference method in performance parameters evaluation. The assay’s performances parameters including analytical specificity (ASp), analytical sensitivity (ASe), diagnostic specificity (DSp), diagnostic sensitivity (DSe), and positive rate between the one-step PCR and nested PCR were compared. Results showed that developed one-step PCR assay demonstrated high ASp for WSSV detection, no cross-reactivity was detected against other 9 common pathogen samples.ASe tests revealed impressive detection limits: using the WSSV standard plasmid as a template, the theoretical minimum detection limit was 1.83 copies/μL, while with mixed DNA from field samples, the limit reached 0.82 fg/μL. Notably, in both scenarios, this method outperformed the WOAH-recommended nested PCR in terms of sensitivity. Comparison of diagnostic performance on 317 field samples showed that the one-step PCR achieved a DSp of 97.01% and a DSe of 95.08%, both superior to the WOAH-recommended nested PCR. In conclusion, this study introduces an innovative one-step PCR method for WSSV detection in P. clarkii, based on the vp19 gene, achieving a remarkable detection limit of 1.83 copies/μL while maintaining simplicity and cost-effectiveness. Outperforming the WOAH-recommended nested PCR, this highly sensitive approach excels in identifying latent infections and facilitating epidemiological investigations. Its ability to detect low-level infections enhances its versatility for both routine screening and research, providing a powerful tool for year-round dynamic monitoring and early warning of WSSV. While acknowledging current limitations, this groundbreaking method lays a solid foundation for future advancements in aquatic pathogen detection. By combining accuracy, sensitivity, and practicality, this research represents a significant step forward in WSSV detection technology, with far-reaching implications for the sustainable development of crayfish aquaculture in both scientific and economic contexts.