The chemical modification of cellulose (Cs)-chitosan (Ch)-electrospun non-woven (ESNW) composite fabrics is described. Since the as-spun Cs/Ch-composite ESNW (Cs:Ch=4:6) deforms in water, an insolublilization procedure using an alkaline-ethanol solution was developed to extend the applications in an aqueous environment. The fine fiber surface was modified with bifunctional isocyanate in order to introduce the -NCO groups on the ESNW surfaces. The -NCO groups were condensed with N,N-(diethyl)ethylene diamine to generate a cationic diethylaminoethyl (DEAE)-ESNW. An enzyme, aminoacylase-I, was immobilized onto the cationic matrix under mild conditions. The immobilized enzyme was subjected to the stereo-specific recognition of N-acetyl-methionines. The results suggest that the chemically modified Cs/Ch-ESNW is a useful support matrix for biological catalysts.