Enzyme immobilization is a promising biotechnology which not only ensures reusability of enzymes and permits easy biocatalyst-product separation, but also improves the storage stability of enzymes. It overcomes many drawbacks of free enzyme, and hence presents significant applications in bioscience and bioengineering. Microspheres composed of biopolymer hydrogel are widely used as carriers for enzyme immobilization. In this study, the regular-sized gelatin microspheres were prepared using the membrane emulsification method, and were subsequently utilized for the encapsulation of yeast alcohol dehydrogenase (YADH). The results indicated that the recycling stability, pH tolerance, and storage stability of the immobilized enzyme are all significantly improved. The immobilized YADH maintained higher relative activity than its free form under extreme acidic conditions down to pH 4.0 (63 vs. 16 %) and alkaline conditions up to pH 10.0 (42 vs. 0 %). Additionally, microspheres of different sizes can be prepared using membranes of different pore sizes. The further experiment established a linear correlation between the average diameter of microspheres and the pore size of membranes. The effects of preparation conditions on the diameter and uniformity of microspheres were also investigated systematically.