Cryptosporidiosis is a significant disease that causes diarrhea in humans and animals with relatively high morbidity and mortality. The present study aimed to adopt a purified and potent antigen for the accurate diagnosis of cryptosporidiosis. A total of 278 animal hosts (60 newborn calves and 218 buffaloes) were used in the current study. Sixty fecal samples were collected from new-born calves aged less than one month raised in the Beni-Suef and Qalyubia governorates. The samples were examined under a microscope after modified Ziehl-Neelsen staining, and Cryptosporidium oocysts were isolated from naturally infected calves. These oocysts were used in mice experimental infection. The oocyst antigen and coproantigen were prepared from the mice feces. The diagnostic efficacy of the two prepared antigens was evaluated using an Enzyme Linked Immunosorbent Assay (ELISA) with experimentally infected mice sera. The crude oocyst antigen proved to have higher diagnostic potential than coproantigen, so, it was chosen for purification using Cyanogen Bromide-activated Sepharose-4B affinity chromatography coupled with rabbit hyperimmune serum raised against oocyst antigen. The affinity purified fraction and its crude Cryptosporidium antigen were evaluated using the ELISA. The resulting purified fraction was 6733 fold increase in binding activity compared with its crude antigen. Characterization of the isolated fraction was performed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blot, and amino acid analysis. SDS-PAGE clarified that the fraction contained three polypeptides of 94.7, 65, and 50 kDa, which were identified as immune-reactive components using a western blot analysis. The isolated fraction exhibited 17 amino acids and was rich in tyrosine, alanine, and phenylalanine. The affinity purified Cryptosporidium oocyst antigen effectively detected Cryptosporidium antibodies in experimentally infected mice sera and naturally infected buffalo sera with a sensitivity of 94.4% and 95.24 %, and a specificity of 100% and 93.33%, respectively. The purified fraction succeeded in diagnosis of cryptosporidiosis in 182 random serum samples collected from buffaloes with an incidence of 57.14 %. In conclusion, the affinity purified fraction of the Cryptosporidium oocyst antigen might be a good diagnostic candidate for cryptosporidiosis diagnosis and seroepidemiological surveillance.