OBJECTIVE： To improve the method for the content determination of vitamin B12 in Multivitamin for injection （12）. METHODS： HPLC was performed on the column of Kromasil 100-5 C18 with mobile phase of water （adjusted to pH4.5 with 10％ diluted phosphoric acid）-acetonitrile （gradient elution） at a flow rate of 1.2 ml/min， the detection wavelength was 361 nm， column temperature was 35 ℃， and injection volume was 50 μl. RESULTS： The linear range of vitamin B12 was 15.03-45.09 ng （r＝0.999 9）； RSDs of precision， stability and reproducibility tests were no lower than 1％； recovery was 99.31％-100.90％ （RSD＝0.42％，n＝9）. CONCLUSIONS： The method is simple， accurate， sensitive， reproducible， and can be used for the content determination of vitamin B12 in Multivitamin for injection （12）.