The aim of this study is to quantify the 146S antigen in foot-and-mouth disease virus (FMDV) inactivated vaccine by size-exclusion chromatography (SEC). The analysis was performed on a TSKgel G4000SWXL column (7.8 mm×30 cm), with a pH 7.2 buffer salt system as the mobile phase. The flow rate was 0.6 mL/min, the injection volume was 100 μL and the detection wavelength was 259 nm. The calibration curve was established by using purified inactivated FMDV (serotype O) 146S antigen; 3 batches of vaccine formulated by inactivated antigen solution were tested to verify the accuracy, reproducibility, specificity and tolerability of the method. At last 16 batches of vaccine were determined by the SEC method. Results showed a good linearity between peak area and concentration of 146S antigen in the range between 0.56 and 67.42 μg/mL (R2=0.996, n=10), and the average recovery rate of 146S antigen in the 3 batches of vaccine formulated in lab were 93.6% (RSD=2.7%, n=3), 102.3% (RSD=2.6%, n=3), and 95.5% (RSD=5.1%, n=3). The method was proved accurate and reliable with good reproducibility (RSD=0.5%, n=6), and applied to determine 16 batches of the commercial FMDV vaccine. According to the above results, the SEC method is high effective for 146S antigen quantify in the inactivated FMDV vaccine and would provide strong support for the vaccine quality control.