Abstract: OBJECTIVE To establish a method to determine the content of fursultiamine hydrochloride, pyridoxamine dihydrochloride and vitamin B₁₂ in compound trivitamin B injection. METHODS C₁₈ column (250 mm×4.6 mm, 5 μm) was adopted, the mobile phase was a mixture of sodium heptanesulfonate solution-acetonitrile-methanol, and elute with linear gradient. The flow rate was 1.0 mL·min^-1 and detection wavelength was 220 nm. The theoretical plate number calculated by the peak of pyridoxamine dihydrochloride was not less than 3 000 and column temperature was 30 ℃. RESULTS The calibtration curve linear of fursultiamine hydrochloride, pyridoxamine dihydrochloride and vitamin B12 were 2.04-20.4 μg, 2.05-20.5 μg and 0.020 6-0.206 μg, respectively, which showed a good linear relationship (r=1.000, 0.999 9, 1.000). The average recoveries were 101.0%, 101.6%, 98.3%, respectively(n=9, RSD=0.50%, 1.40%, 0.30%). CONCLUSION The method is simple, accurate, repeatable and can be applied to determine the content of fursultiamine hydrochloride, pyridoxamine dihydrochloride and vitamin B₁₂ in compound trivitamin B injection.