The fermentation medium of alcohol dehydrogenase produced from acetobacter aceti was optimized by single factor experiments and orthogonal experiments to increase the strains’ activity of producing alcohol dehydrogenase. The results showed that the optimal formula was glucose 1%, yeast extract 2%, ethanol 3%, MgSO4 0.05 %, KH2PO4 0.45%. Under the above condition, the enzyme activity was 0.423U/mL after fermentation for 3 days.