In this work, trypsin was incorporated into poly(L-lactide) (PLLA) using electrospinning technology to obtain the bioactive nanofibers for gelatin digestion. In order to improve the stability and the efficiency of trypsin incorporated PLLA nanofibers (NF-TR), glutaraldehyde was used to crosslink trypsin molecules onto fibers surface (CL-TR). CL-TR showed better gelatin digestion efficiency than NF-TR in rigorous conditions, such as at high temperatures of 40 degrees C and 50 degrees C, in the presence of alcohol or dimethyl-sulfoxide, and at various pH values. CL-TR also exhibited higher stability than NF-TR. The improved efficiency and stability of CL-TR can be explained by the crosslinked trypsin on the surface of PLLA nanofibers effectively inhibited the denaturation, autolysis, and leaching of trypsin. The LC-MS/MS results showed that gelatin sample would be digested into amino acids and/or oligomers. The trypsin incorporation approach is anticipated to be employed not only for protein digestion but also for various other fields where the poor enzyme stability presently hampers the practical applications of enzymes.