Aquatic prefabricated dishes are widely favored for their convenience; however, they are susceptible to contamination by foodborne pathogens during processing and storage, posing a threat to food safety. To rapidly and accurately screen such risks, a multiplex quantitative real-time polymerase chain reaction detection system was developed in this study. Specific primers and TaqMan probes were designed based on the unique target sequences of Vibrio parahaemolyticus, Listeria monocytogenes, and Salmonella spp. In pure culture tests, the minimum detection limits for V. parahaemolyticus, L. monocytogenes, and Salmonella spp. were 8.67×101, 3.47×102, and 6.56×103 CFU/mL, respectively. A strong linear relationship was observed between the logarithmic bacterial concentrations and Cq values, with correlation coefficients (R2) exceeding 0.98. Within the concentration range of 108~101 CFU/mL, the coefficient of variation from three replicate amplifications was within 5.08%. In artificially spiked samples, after 12 h of enrichment, the detection limits improved to 6.30×100, 6.13×102, and 2.44×101 CFU/mL for the three pathogens, respectively. The accuracy of the system was validated by testing 60 real aquatic prefabricated dish samples, achieving a 100% concordance with traditional culture methods. This detection system was highly specific, sensitive, stable, and resistant to interference, enabling rapid and precise identification of the three foodborne pathogens in aquatic prefabricated dishes. An efficient tool was thus provided for monitoring foodborne pathogens in aquatic prefabricated dishes, contributing significantly to consumer health protection.