The para-nitrobenzyl esterase (PNBE) was successfully immobilized onto electrospun polystyrene/poly(styrene-co-maleic anhydride) (PS/PSMA) nanofibers as cross-linked enzyme aggregates (CLEA). The maximum amount of PNBE immobilized on the PS-PSMA nanofibers was determined to be 56.4 mg/g of nanofibers. The variation of pH and temperature has been observed to affect the enzymatic activity of the free and immobilized PNBE-CLEA. The immobilized PNBE-CLEA has been observed to show improved stability as compared to the native one. PNBE-CLEA has been found to retain more than 63% activity after 60 consecutive reuses. Finally, the immobilized PNBE-CLEA presented a high decomposition efficiency of cephalexin tosylate PNB ester. The durability determination of PNBE-CLEA immobilized nanofibers showed 70% of de-esterification after 10 cycles. These results demonstrate that PNBE-CLEA immobilized nanofiber could be used as an economical and environment friendly system for the catalytic process of para-nitrobenzyl esters in pharmaceutical synthesis.