The crystallization is one of key step of the penicillin production , it generally adopts the method of butanol decompressed azeotropic distillation. Through the research and analysis on crystalline supersaturation, the final crystallization water content and nature of RB,the results showed that the crystalline process control was optimial when pH of RB was 6.4, the potency of RB- butanol solution was 300 000 units per millilitre, the final crystallization water conten was from 0.6% to 1.2%,thereby improving the product quality and yield and favoring the process of three-in-one.