Foot-and-mouth disease (FMD) remains one of the most economically important transboundary animal diseases affecting livestock production worldwide. Despite systematic vaccination under the National Animal Disease Control Programme (NADCP) in India, Foot-and-Mouth Disease Virus (FMDV) serotypes O, A, and Asia-1 continue to circulate in endemic regions. Post-vaccination serological surveillance is an important component of disease control programmes for evaluating level of immunity in vaccinated population and identifying gaps in acquiring herd immunity. The present study aimed to evaluate serotype-specific antibody responses against FMDV serotypes O, A, and Asia-1 in bovines of Western Uttar Pradesh using Solid Phase Competitive Enzyme-Linked Immunosorbent Assay (SPCE), compare district-wise herd immunity levels, and assess the serological status of bovines sampled before and after routine vaccination under field conditions. A cross-sectional serological investigation was conducted during 2025–26 across sixteen districts of Western Uttar Pradesh. A total of 827 bovine serum samples, comprising 333 pre-vaccination and 494 post-vaccination samples collected under field conditions, were analysed using SPCE for detection of antibodies against FMDV structural proteins. Protective antibody titres were defined as ≥1.8 log10 based on the manufacturer's recommendations and established seromonitoring guidelines. Seropositivity was expressed as percentages with 95% confidence intervals, and differences between pre- and post-vaccination groups were evaluated using Pearson's Chi-square test. Animals sampled after routine vaccination exhibited substantially higher serotype-specific antibody prevalence than those sampled before vaccination. Protective antibody prevalence was 67.08% (95% CI: 62.9–71.2%) versus 28.37% (95% CI: 23.4–33.1%) for serotype O, 65.36% (95% CI: 61.2–69.6%) versus 25.32% (95% CI: 20.6–29.9%) for serotype A, and 65.18% (95% CI: 61.0–69.4%) versus 24.43% (95% CI: 19.7–28.9%) for serotype Asia-1 (P < 0.001 for all comparisons). Serotype O showed the highest post-vaccination seropositivity. Considerable geographical variation in antibody prevalence was observed among districts, with Baghpat, Kasganj, and Mainpuri demonstrating comparatively higher levels of protective antibodies than several other districts. Overall mean seropositivity increased from 26.04% in the pre-vaccination group to 65.87% in the post-vaccination group; however, this level remained below the operational herd immunity target commonly recommended for interruption of FMDV transmission in endemic settings. The findings indicate that bovines sampled following routine vaccination exhibited substantially higher serotype-specific antibody prevalence than animals sampled before vaccination, supporting the immunogenicity of the trivalent FMD vaccine under field conditions. Nevertheless, persistent immunity gaps and marked district-wise heterogeneity highlight the need to strengthen vaccination coverage, targeted booster immunisation, and continuous post-vaccination seromonitoring. SPCE proved to be a practical tool for large-scale assessment of population-level immunity and may support evidence-based decision-making for progressive control of FMD in endemic regions.