Recombinant human serum albumin （rHSA）has being used in the production of high-dose biological products, which poses a great challenge to the downstream separation and purification technology. Hydrophobic chromatography separation is achieved by using the interaction between hydrophobic groups of sample components and hydrophobic groups of media, which can effectively remove degradants in the purification of rHSA. Phenyl Sepharose 6FF (HS) hydrophobic separation medium from GE Company was selected to separate and purify rHSA intermediates, and the process control conditions were optimized. The experimental results show that the best removal efficiency and protein yield are obtained by using 50 mmol/L PB + 0.1 mol/L NaCl buffer and controlling pH value of 6.0.