Ethanol precipitation is an important purification process affecting the safety and efficacy of botanical injections. In this paper, the second ethanol precipitation of Guanxinning injection was optimized with response surface methodology as a sample to represent the principles of indicator selection. The influences of three factors, including dry matter content of concentrated supernatant (DMCCS), refrigeration temperature, and the mass ratio of ethanol and concentrated supernatant (E/CS ratio) were explained based on the precipitation mechanism. To characterize the effects of ethanol precipitation on drug safety, drug efficacy, and drug quality control, phenolic compound recovery and dry matter removal were selected as optimization criteria. In the optimization, E/CS ratio was set to 3.60. The optimum operating conditions were found at refrigeration temperature of 18.66 degrees C and DMCCS of 0.58. Most of the experimental results at optimum operating conditions agreed well with the predicted results.