To develop a rapid differentiation and quantitative method for detecting the pili antigen of E. coli K88,K99 and 987P strain,positive serum of rabbit was prepared with purified E. coli K88,K99 and 987P pili antigen and their AGP titers were 1∶32,1∶32 and 1∶128,respectively. Through sensitizing the Aldehydated sheep erythrocyte with the positive serum IgG,the optimal IgG concentrations in K88,K99 and 987P positive sera were determined at 40 μg/mL,160 μg/mL and 80 μg/mL,respectively. The same batch of K88,K99 and 987P pili antigens were detected three times using the same method and the RIHA titer of K88,K99 and 987P samples were 1∶1 600,1∶1 600 and 1∶800,respectively. The results showed that the detecting method had good specificity and reproducibility and could be used for differentiation and quantitative detection of pili antigen of E. coli K88,K99 and 987P strain.