A high performance liquid chromatographic method was developed for the simultaneous separation and determination of vanillin and o vanillin. Influences of mobile phase composition and column temperature on the separation were investigated. Under the condition of 5% acetic acid-acetonitrile （60∶40,by volume） as mobile phase at a flow rate of 10 mL/min and 25 ℃,the vanillin and o vanillin were separated in less than 5 min. The calibration curves of vanillin and o vanillin showed good linearities in the range of 10-240 mg/L with both correlation coefficients of 0999 7. The limits of detection（S/N=3） for vanillin and o vanillin were 006 mg/L and 002 mg/L,respectively. The average recoveries at three spiked levels were in the range of 99% -102% with relative standard deviations of 02% - 06%. The method was successfully used for the determination of vanillin and o vanillin in real samples with satisfactory results.