The present invention provides a method for preparing biodiesel, comprising: adding oil and fat, short-chain alcohol, water, and liquid lipase into a single-stage or multi-stage enzyme reactor; then separating the reaction fluid into an enzyme-containing heavy phase and a light phase; recovering and reusing the enzyme in the heavy phase; using the light phase for subsequent conversion by immobilized lipase; flowing the light phase and the short-chain alcohol into single-stage or multi-stage enzyme reactor containing an immobilized lipase; performing online dehydration during the whole reaction process or part of the reaction process. In the method of the invention for preparing biodiesel, no preprocessing is required for the oil and fat feedstock in the earlier stage of catalysis process by liquid lipase, and the conversion ratio from oil and fat to biodiesel can reach more than 90%; in the later stage of catalysis process by immobilized lipase, by introducing an online dehydration during the whole process or part of the reaction process, the yield of biodiesel can exceed 98%, and the acid value of the product can be less than 0.5 mg KOH per gram of oil. The method thus has good economic and environmental benefits.