Low stability was one of the biggest problems during traditional purification process of recombinant hepatitis B surface antigen (rHBsAg). A combination process of silica gel adsorption/desorption and hydrophobic interaction chromatography was proposed in this work. The effect of pH on HBsAg stability was studied from the particle integrity of view using static light scattering, fluorescence spectrum and dynamic light scattering, respectively. HBsAg was purified by silica gel adsorption/desorption using response surface methodology for experimental design, followed by hydrophobic interaction chromatography, and both its morphology and particle integrity were studied. In acidic solution, the electrostatic repulsion of HBsAg particles decreased leading to aggregation when pH of solution was close to the isoelectric point of HBsAg. In alkaline solution, hydrophobic patches inside HBsAg particles were likely to be exposed leading to disaggregation. The silica gel adsorption/desorption process was optimized. When HBsAg activity recovery was taken as the response value, the optimum operating condition was as follows: adsorption pH was 7.43, desorption pH was 10.48 and desorption temperature was 55.4℃, and HBsAg activity recovery was 39.1%. When purification fold was taken as the response value, the optimum operating condition was as follows: adsorption pH was 7.16, desorption pH was 10.52 and desorption temperature was 55.1℃, and purification fold was 1.90. After hydrophobic interaction chromatography, HBsAg activity recovery was 49.73% and the particle integrity was 85.79% respectively. Compared with conventional hydrophobic interaction chromatography, the efficiency of HBsAg purification was improved greatly using this combination method, etc., HBsAg activity recovery increased by 31.99%, the particle integrity by 20.90%, and the particle stability by 22.93%, respectively. It provided a new idea for both high efficient recombinant HBsAg purification and improvement of antigen particle integrity.