We investigate the immobilization of trypsin on chitosan nonwoven using glutaraldehyde (GA). The conditions for trypsin on chitosan nonwoven and GA cross-linking were optimized depending on different conditions. The order of GA cross-linking was determined by the activity of immobilized trypsin. The characteristics of chitosan nonwoven were examined by Fourier-transform infrared (FT-IR) and surface morphology analyses (SEM). Results showed that the optimal treatment conditions for trypsin on chitosan nonwoven were as follows: pH 8.5; temperature $37^{circ}C$; trypsin concentration 15% (o.w.f); and treatment time 60 min. Those for GA cross-linking were: pH 10.0; GA concentration 3% (v/v); and treatment time 120 min. FT-IR analysis showed that GA was cross-linked on chitosan nonwoven. The SEM analysis also showed that trypsin was immobilized on chitosan nonwoven.