Objective: The molecular biological test has the advantage of fast and sensitive, which can effectively make up for the defects of the traditional microbial test method. Methods: According to the conserved sequences of the V3~V4 region of the virulence gene pcrL and the universal gene 16S rRNA of Pseudomonas aeruginosa in GB 8538—2022, primers and probes were designed and synthesized, and a real-time fluorescent PCR kit was established in this study. Results: The results showed that the common standard reserve strain 16S rRNA was amplified, and the virulence gene pcrL was only effective for nucleic acid detection of Pseudomonas aeruginosa standard reserve strain, and obvious "S" type amplification curve could be observed, while no amplification curve was found for common food-borne pathogens in this study. Gene pcrL still has amplification curve, when the concentration of bacterial solution was less than 10 CFU/mL, the detection range was 10~10⁷ CFU/mL, and the detection limit was 10 CFU/mL. The Ct mean value of virulence gene pcrL under different bacterial solution concentrations ranged from 18.0 to 38.6. The results showed that this method was highly sensitive to the detection of the virulence factor pcrL carried by Pseudomonas aeruginosa. In the past three years, the unqualified Pseudomonas aeruginosa samples in 14 counties and cities of Hunan Province were further detected, and the coincidence rate reached 100%. Conclusion: This method is highly sensitive to detect the virulence factor pcrL carried by Pseudomonas aeruginosa. It has the advantages of simple operation, strong specificity, high sensitivity and good practicability.