Based on gold label silver stain and coupled with multiplex asymmetric polymerase chain reaction (PCR) analysis, we developed the visual DNA microarray for simultaneous, sensitive, and specific detection of human immunodeficiency virus type-1 (HIV-1) and Treponema pallidum. The 5'-end amino-modified oligonucleotides were immobilized on glass surface, which were used as the capturing probes to bind the complement biotinylated target DNA. The gold-conjugated streptavidins were introduced to the microarray for specific binding to biotin. The black image of microarray spots, which were the result from the precipitation of silver onto nanogold particles and bound to streptavidins, was visualized and accounted as the detection of biotinylated target DNA. Multiplex asymmetric PCR products of HIV-1 and T. pallidum and Bacillus subtilis (used as positive control) were performed for preparing the abundant biotinylated single-stranded target DNA of which could affect detection efficiency and sensitivity of hybridization on microarray. One hundred sixty-nine clinical samples of HIV-1 and T. pallidum from infected patients were tested using the homemade DNA microarrays. The results were identical to those shown in the assays of ELISA and fluorescence quantitative real-time PCR. Our results demonstrate that we have developed the visual gene detection technique, which is of high sensitivity and specificity; it may have potential in clinical applications.