The invention relates to a process for enzymatic preparation of chiral 3-hydroxytetrahydrofuran and an alcohol dehydrogenase mutant. The process comprises the a step of, with 3-ketotetrahydrofuran as a substrate, subjecting the substrate, the alcohol dehydrogenase mutant, a cofactor and a cofactor regeneration system to a biological catalysis reaction in a buffer system so as to produce chiral 3-hydroxytetrahydrofuran, wherein for alcohol dehydrogenase used by the alcohol dehydrogenase mutant, the wild gene sequence of alcohol dehydrogenase is derived from thermophilic bacteria, the base sequence is as shown in SEQ ID No. 1, and the amino acid sequence is as shown in SEQ ID No. 2. Compared with chemical methods, the process using the alcohol dehydrogenase mutant for preparation of chiral 3-hydroxytetrahydrofuran has the advantages of small environmental pollution, high efficiency, high optical selectivity; and compared with enzyme resolution methods, the process provided by the invention has higher purity and conversion rate and has ee value and conversion rate of both 99%.