An optimized method has been established to simultaneously detect multiple antibodies of HIV, TP and HCV in the serum by the way of microarray. Chimeric antigens of HIV, TP and HCV are covalently bound to the surface of glass slide; serum samples are diluted and added to the well of microarray, unbound antibodies are washed away after incubation, and Cy3 labeled second antibody is added to the well followed by incubation and washing. The microarray is scanned by laser confocal scanner, and the resulting images are treated by the software of Imagene (produced by Bio-discover Co.). The data obtained is then treated by the software produced by own; the evaluated result of each sample is finally gained automatically by the Cutoff value of each antigen. 400 normal serum sample are detected and Cutoff value of each antigen are determined. National panels of HIV, TP and HCV had also been detected by this system and the results were compared with those detected by ELISA method. The coordinate ratios of positive and negative samples of HIV panel are both 100%(20/20); these kinds of the coordinate ratios of positive and negative samples of HCV are both 95%(38/40) and the coordinate ratios of positive and negative samples of TP are 100%(10/10) and 100%(20/20) respectively. This result is highly in accord with that of ELISA method.