Poly(vinyl alcohol), PVA, membrane was prepared by crosslinking reaction initiated by UV irradiation in the presence of sodium benzoate and benzoic acid, Penicillinase was immobilized after the PVA membrane had been activated with cyanuric chloride. The penicillinase-immobilized PVA membrane was then coupled with a pH glass electrode and used as a penicillin sensor. The effective diffusion coefficient of hydrogen ions through the penicillinase-immobilized PVA membrane was determined to be about 1.4 x 10(-6) cm(2)/sec. This value is about two orders of magnitude higher than that obtained from the penicillinase-BSA (bovine serum albumin) membrane, Increasing the buffer strength increased the mass transfer rate of hydrogen ions, but decreased the sensitivity of the enzyme electrode, The higher buffer strength nevertheless increased the linear analytical range of the penicillin electrode, These results demonstrate the applicability of using PVA hydrogel as an excellent enzyme immobilized matrix for biosensor.