The method of HPLC for determination of methionine and vitamin B1 in the injection was established. A ODS C18 column (4.6 mm × 250 mm, 5 μm) was used with the mobile phase of buffer [sodium heptanesulfonate dissolved in sodium phosphate solution (38 g/per 1000 mL, adjusting pH to 2.1 by phosphoric acid)]-acetonitrile (94:6), flow rate was 1.0 mL/min, and detection wavelength was at 210 nm. Methionine and vitamin B1 had linear ranges of 0.60-1.40 g/mL and 0.06-0.14 mg/mL, resp. The average recoveries of methionine and vitamin B1 were 100.49% and 99.95% with RSD of 0.62% and 0.48%, resp. The method was convenient, accurate and reliable for determination of methionine and vitamin B1 in the injection.