The study aimed to serologically detect the prevalence of Sarcocystis infection in buffaloes using the indirect enzyme-linked immunosorbent assay (ELISA) for the first time in Iraq. An overall 184 buffaloes of different ages and sexes were selected randomly from the regions of Wasit province (Iraq) from March to June (2022), and subjected to draining of venous blood under aseptic conditions. After centrifugation, the obtained sera were examined by indirect ELISA. Totally, 33.15% of study animals were positive for IgG antibodies. According to their concentrations, the ODs level showed a significant increase (P<0.0186) in mild infection (63.93%) when compared to moderate (27.87%) and strong (8.2%) infections. Subsequently, values of mild, moderate, and strong infections were 0.330 ± 0.01, 0.554 ± 0.019 and 0.912 ± 0.031 nm, respectively. Regarding age and sex factors, significantly higher positivity (P<0.0469) was shown in buffaloes aged >10 years old (57.89%); while the lowest was seen in those <1 year (13.64%). Subsequently, older buffaloes appeared significantly (P<0.0192) at higher risk (2.1685) when compared to other age groups. Females recorded an obvious increase (P<0.05) in positivity (35.95%) and risk (1.8569) in comparison with males; 19.35% and 0.5385, respectively. The results indicate that ELISA is of great value in the diagnosis of sarcocystosis.