Human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health. Based on multiplex asymmetrical PCR and coupled with gold labelled silver stain (GLSS), we developed the visual DNA microarray for sensitive and specific detection of these two viruses. Capturing probes of 5'-end-amino-modified oligonucleotides were immobilized on glass surface to bind the complement biotinylated target DNA. The Au-streptavidin probe was introduced to the microarray for specific binding to biotin. Black images of microarray spots which result from the precipitation of silver onto Au-streptavidin probes, were visualized by naked eyes. In order to improve the efficiency of microarray hybridization, triplex asymmetrical PCR of HIV-1, HCV and Human enterovirus 71 (EV-71, used as positive control) were performed to prepare abundant biotinylated single-stranded target DNA. The sensitivity of visual DNA microarray (10(3) copies/ml) was higher than conventional PCR (10(4) copies/ml) and was identical to FQ-PCR (10(3) copies/ml). Total 152 blood samples containing the two viruses were tested using the DNA microarray and fluorescence quantitative real-time PCR (FQ-PCR). The results were identical (P > 0.05). So this system has high sensitivity and may have potential in clinical applications.