Abstract: There are some problems in the teaching experiment of yeast alcohol dehydrogenase (YADH) extraction and purification. In order to obtain YADH with high purity, high activity, and high recovery, single factor experiment was applied in this study to optimize the separation and purification process of enzyme protein, including hot extraction, thermal denaturation precipitation, and organic solvent precipitation. This will improve the extraction and purification efficiency of YADH. The results showed that the best effect of separation and purification was achieved when the solid-liquid ratio was 1 : 6, the crude extraction temperature was 25 ℃, the crude extraction time was 1.5 h, the thermal denaturation temperature was 65 ℃, the thermal denaturation time was 15 min, the organic solvent precipitant was ethanol, the adding proportion of precipitated impure protein was 0.5 times of the supernatant, and the adding proportion of precipitated enzyme protein was 0.45 times of the supernatant. Through the optimization of the above conditions, the effect of teaching experiment is improved. It is also helpful to exercise students’ practical skills, innovative thinking, and scientific research abilities.