Abstract: Objective: To establish a method for the determination of nine vitamin B components in vitamin B tablets by high performance liquid chromatography-diode array detector (HPLC-DAD). Method: Folic acid was ultrasonically extracted with 0.3% ammonia solution, biotin, vitamin B₁₂ and pantothenate acid were ultrasonically extracted with water, the vitamins B₁, B₂, B₆, niacin and nicotinamide were ultrasonically extracted by 0.1% hydrochloric acid, separated by C₁₈ column with methanol-0.1 mol/L potassium dihydrogen phosphate solution as the mobile phase for gradient elution. Pantothenate acid and biotin were detected at the wavelength of 205 nm, vitamins B₁, B₂, niacin and nicotinamide were detected at the wavelength of 256 nm, vitamins B₆ and folic acid were detected at the wavelength of 287 nm, vitamins B₁₂ was detected at the wavelength of 361 nm. Results: The results showed that the nine components could be separated well, and the linear relationship was good in their linear range. The average recoveries were 91.8%~106.9%, RSD were 0.2%~4.8%, and the detection limit of the method were 0.05~4.7 mg/kg. Conclusion: This method is rapid, simple, accurate and repeatable. It can quickly complete the determination of nine vitamin B components in health food vitamin B tablets and reduce the cost.