To achieve rapid and simultaneous detection of two key virulence genes in Aeromonas hydrophila, act and lip, in aquatic products, double nucleic acid test strips based on polymerase chain reaction coupled with immunocolloidal gold technology were developed. The optimal labeling amounts for streptavidin, C-line, T1, and T2 detection lines were determined, validated, and evaluated for specificity, sensitivity, reproducibility, and stability, followed by testing using artificial simulated and actual samples. The optimal labeling amounts for streptavidin, C-line, T1, and T2 detection lines were 5 μL and 2, 0.275, and 0.2 mg•mL-1, respectively. In terms of specificity, only A. hydrophila was positive, and the other bacteria were negative. In terms of sensitivity, the lowest limit of detection of the double nucleic acid strips was 10-2 ng•μL-1. In terms of reproducibility, the results were verified by operators from three different laboratories, yielding consistent results. In terms of stability, the double nucleic acid test strips were evaluated at 6, 9, and 12 months and exhibited good stability. Testing using both artificial simulated and actual samples showed that this method can rapidly and accurately detect A. hydrophila. The double nucleic acid test strip method established in this study can rapidly and simultaneously detect act and lip virulence genes in A. hydrophila with high specificity, high sensitivity, good reproducibility, good stability, short detection times, and visualization of results. These findings provide a theoretical basis and new directions for aquatic food safety assurance.