Theileria are tick-borne apicomplexan parasites, which mainly infect ruminants in tropical and subtropical regions of the world. The present study was directed to investigate the serological methods for the diagnosis of theileriosis in crossbred cattle. Blood samples (n = 176) were collected from the regional cattle populations of Bihar state situated at the Gangetic plains of India. Microscopic examination of blood smears from the cattle revealed the presence of tick-borne infectious organisms (Theileria and Anaplasma) in the region. PCR-based detection of Tams1 (Theileria annulata merozoite surface antigen) gene and the sequencing of 18S rRNA amplicon from the blood samples confirmed T. annulata as the primary causative agent of theileriosis in cattle of the Bihar region. Similarly, the amplification of the msp5 gene confirmed Anaplasma marginale infection. For the large-scale epidemiological investigation, sporozoite and macroschizont (spm2) partial gene from T. annulata was cloned in pET-28a (+) vector and overexpressed in E. coli BL21 cells. Overexpressed recombinant-Spm2 (43 kDa) was purified by Ni-NTA affinity chromatography and was used for immunodetection of theileriosis in cattle serum samples. Sequence analysis of the cloned partial spm2 gene in this study showed multiple SNPs (single nucleotide polymorphisms) in T. annulata. Recombinant-Spm2 antigen was explicitly recognised by the immunoglobulins (IgG) of the cattle naturally infected with Theileria. Further, an indirect enzyme-linked immunosorbent assay (ELISA) was developed using partial r-Spm2 antigen that exhibited high sensitivity (100 %) and specificity (90.9 %). Thus, this study suggests that partial r-Spm2 can be used as a diagnostic antigen for seroepidemiological studies of T. annulata infection in crossbred cattle.