Objective To examine whether optimization of enzyme immunoassay reaction conditions improves the sensitivity of antibody detection for hand, foot and mouth disease (HFMD). Methods Serum samples from 100 patients with HFMD confirmed by nucleic acid testing from May 2022 to May 2025 were selected. Reaction time, incubation temperature, enzyme concentration, and antibody dilution were optimized by a single-factor gradient approach. Standard curves were established under different conditions, and assay sensitivity, specificity, linearity, and repeatability were evaluated. Results Assay performance differed significantly across reaction conditions (all P＜0.05). The best results were obtained with a reaction time of 45 min, an incubation temperature of 37 ℃, an enzyme concentration of 0.5 μg/mL, and an antibody dilution of 1:2000. Under these conditions, sensitivity reached 89.50% and specificity reached 92.00%. The standard curve showed excellent linearity (R2＝0.995), background signal was reduced, and repeatability was acceptable (RSD＜3%). Conclusion Optimization of key reaction parameters improved the analytical performance of the enzyme immunoassay for HFMD antibody detection, particularly in terms of sensitivity, specificity, linearity, and stability.