To obtain an efficient method for determination of total vitamin C (VC) in bee pollen, high-performance liquid chromatography (HPLC) was employed and investigated for its stability, precision and repeatability in qualitative and quantitative analysis of total VC in bee pollen. As the results, total VC content and chromatogram peak area presented good linear correlation. The recoveries were 95.71%~98.60% with RSD 1.05%. The RSD of the stability and precision experiments were 2.12% and 1.1% respectively. Total VC content in rape bee pollen was 36.99mg/100g in average, and its RSD was 1.43%. In conclusion, HPLC can be used as a simple and fast method for qualitative and quantitative analysis of total VC in bee pollen.