Foot and mouth disease (FMD), caused by apthovirus of picornaviridae family, is a highly contagious disease leading to severe economic losses to dairy industries in the affected countries. A cross sectional study was conducted to estimate the prevalence of circulating antibodies against non-structural proteins of FMD virus in the livestock in Uttar Pradesh. A total of 400 sera samples from the animals (200 cattle and 200 buffaloes) were tested for the presence of antibodies against r3AB3 non-structural protein (NSP) of FMDV using ELISA. The overall prevalence of anti-r3AB3 antibodies in bovine samples tested by the r3AB3-ELISA was 27.75%. No significant difference in prevalence of anti- r3AB3 antibodies was found in species and sex. At age level seroprevalence varied significantly among animals of <6 months (10%), 6 Months to 1 Year (13.33%), 1-3 Years of age (28.00%) and > 3 years of age (34.71%). At region-level, the significantly higher prevalence was recorded in Bareilly (47.50%) followed by Moradabad (40.00%), Bijnor (37.50%), Amroha (7.50%) and Rampur (6.25%). From the study, it can be concluded that natural infection of FMDV is widespread in the area and further studies using large number of samples including the molecular typing of FMD virus isolated from outbreaks and serum neutralization studies should be employed to develop an effective control program in India.