Abstract: This study was aimed to establish a HPLC approach for the quantitative analysis of campesterol, stigmasterol and β-sitosterol in phytosterol. In HPLC analysis, the chromatographic separation was achieved on a Waters Symmetry C₁₈column (4.6 mm×250 mm, 5 μm) with a mobile phase composed of acetonitrile and water at a flow rate of 1.0 mL/min. In addition, detection wavelength and column temperature were set at 208 nm and 30℃, respectively. The results showed that three sterols reached the baseline separation and the concentration ranges of campsterol, stigmasterol and β-sitosterol were 2.52~50.30, 5.08~101.60 and 5.10~102.00 μg/mL respectively. It indicated a good linear relationship and the correlation coefficients of three sterols was 0.9971, 0.9989, 0.9991.The limit of detection was 2.5 μg/mL, the intra-day precision was between 0.06%~3.06% and the inter-day precision was between 1.56%~6.61%, the recovery of the three components was 92.74%~106.25%. This method could accurately measure campesterol, stigmasterol and β-sitosterol in phytosterols from 4 kinds of production places, the content of 3 individual phytosterols from deodorizer distillate and tall oil had great differences. The content of campesterol and stigmasterol in phytosterol was 163.80~251.23 mg/g and 134.89~235.04 mg/g, much higher than content in wood sterol. The content of β-sitosterol in wood sterol was (685.10±7.55) mg/g, which was higer than phytosterol. It could be concluded that this was a better method for quality assessment of campesterol, stigmasterol and β-sitosterol in phytosterol.