Abstract: A method was developed for the determination of vitamin B_1,B_2,B_3,B_6,B_12 in yak milk by reversed phase high performance liquid chromatography method. After derosination by centrifugation and deproteinization with perchloric acid,the sample was separated on a C_18 column( 250 mm × 4.6 mm,i.d.5 μm) at 25 ℃. A gradient programme was carried out using 0.023 mol/L H_3PO_4( pH3.54) and methyl alcohol as elution solvent at a flow rate of 0.5 mL/min. The response signal was detected at 270 nm.The results showed that vitamin B_1,B_2,B_3,B_6,B_12 could be separated within 20 min.The calibration curves were linear in the ranges of 0.1 ~ 50.0 mg/L for vitamin B_2,B_3,and 0.5~250.0 mg/L for vitamin B_1,B_6,B_12 with correlation coefficient of 0.999. The spiked recoveries of vitamin B in yak milk were in the range of 90.6% ~ 98.4% with the relative standard deviations( RSDs) of 2.0% ~4.3%. The limits of detection( LODs) of five vitamin B in yak milk were in the range of 0.01~0.09 mg/L,the limits of quantitation( LOQs) were 0.03 ~ 0.29 mg/L. The developed method was sensitive and precise,which could meet the demands of rapid analysis.