[Objective] To develop a HPLC for determination of purity of surface antigen of recombinant hepatitis B vaccine (Hansenula polymorpha). [Method] The TSK gel G5000 PW column was used and a series of experiment parameters such as mobile phase, flow rate, column temperature and pH for determination the purity of recombinant hepatitis B vaccine were optimized and the optimal conditions were chosen, the established method was compared with the method by TSK gel G3000 SW chromatog. column. [Result] The established optimal conditions were 0.01 mmol/L PB (Phosphate Buffered) as mobile phase, 0.6 mL/min as flow rate, room temperature (20°C) as column temperature, 7.0 as pH, and 280 nm as detection wavelength. The retention time of surface antigen (HBsAg) in TSK gel G5000 PW and TSK gel G3000 SW were 8.5 min, 17.5 min and the testing time were 20 min, 40 min. [Conclusion] The developed method showed high specificity, repeatability, efficiency, timesaving and it can be used for determination the purity of HBsAg.