A facile enzymatic one-pot multicomponent synthesis of 4-thiazolidinones was developed. The trypsin from porcine pancreas displayed great catalytic activity to promote the reaction of aldehyde, amine and mercaptoacetic acid with high yields showing a broad substrate specificity in this reaction. This trypsin-catalyzed multicomponent conversion method provided a novel strategy to synthesize thiazolidinones and expanded the application of trypsin in organic synthesis. .