Disclosed is a process for preparing biodiesel from renewable oil and fat catalyzed by lipase with online dehydration. The method uses a short chain alcohol ROH as an acyl-acceptor, and utilizes a lipase as the catalyst to catalyze transesterification reaction of oil and fat feedstock with said short chain alcohol, so as to synthesize biodiesel; and online dehydration with a membrane or a molecular sieve is carried out during the whole process or part of the reaction process. Such online dehydration process greatly simplifies the operation procedure, and provides good economic and environmental benefits.