An affinity purified Babesia bigemina piroplasm antigen was successfully employed in a Dot-ELISA for detection of antibodies in buffaloes and cattle. A low degree of cross-reactivity was seen against putative B. bigemina (Mexican) and B. bovis undiluted reference serum and no cross-reactivity was observed in serum dilutions of 1:10 and above. Theileria annulata did not show any reactivity with B. bigemina. The assay detected antibodies at 7, 14 or 21 days post-experimental infection in bovine calves with 37.5, 87.5 and 100% seroreactivity, respectively. The incidence of B. bigemina infection was found highest in cattle (19.91) as against 7.4% in buffaloes out of 486 sera samples, examined from Boophilus microplus infested areas from Orissa, Rajasthan and Uttar Pradesh States.