Abstract: Direct ELISA (D-ELISA) with polystyrene balls has been established to dectect PRV antigen. In the D-ELISA, the working concentrations of the conjugates, P3-HRP and P10-HRP, were 2.61 and 2.85μg /ml of Ig, respectively. With the two conjugates and their mixture, 5.63μg/ml of PRV protein (crude extract), or even lower, could be detected by the D-ELISA. The PRV antigen in the mice infected with PRV was checked by the D-ELISA with mixture, and the results indicated that positive rate of PRV antigen was 98.0% by measuring the five kinds of samples: liver, lung, spleen, kidney and submaxillary gland-subaural gland mixture. The two gland mixture had the highest positive rate (87.0%), in average and then both of lung and spleen had similla rates, 51.8-71.4%, liver 26.3-61.3%, and kidney 28.6-95.0%. The positive reaction of the two gland mixture was singnificantly different from the negative reaction of the control samples, and could be distinguished with the naked eyes. These results showed that the D-ELISA with polystyrene balls would be a highly sensitive, specific, rapid and simple technique for the clinical diagnosis of pseudorabies.