Vibrio parahaemolyticus-induced food poisoning is frequently reported, highlighting the necessity to develop a simple, rapid, large-scale on-site detection method to ensure food safety and avoid financial losses. Time-resolved fluorescence lateral flow immunoassay (TRF-LFIA) was successfully developed for the rapid detection of Vibrio parahaemolyticus, using europium (III) nanoparticles (EuNPs) to label V. parahaemolyticus monoclonal antibodies, forming microsphere-antibody conjugations for use as specific immunofluorescent probes. The intensity of fluorescence signals emitted by the combination of the probes and the target pathogenic bacteria could be detected to achieve quantitative detection of the target pathogenic bacteria. The TRF-LFIA has a sensitivity of 8.2×102 CFU/mL, a visual detection limit of 1.2×104 CFU/mL, a linear range of 1.8×103~1.8×107 CFU/mL, a recovery rate range of 84.25%~105.13%, and a coefficient of the variation range of 2.56%~9.14%. There was no cross-reaction to the other seven major foodborne pathogenic strains. The TRF-LFIA established in this study has high sensitivity, allowing for simple and rapid operation, and good repeatability and specificity, providing a powerful detection tool for rapid on-site detection of V. parahaemolyticus.