Chemically cross-linked PVA films are permeable matrices for the fabrication of biosensors. PVA provides an attractive immobilisation method as it preserves the enzymatic activity. Penicillinase (P'nase) was cross-linked with poly(vinyl alcohol) (PVA) and bovine serum albumin (BSA). The optimum conditions for the of BSA-PVA-P'nase film were: 2.5% w/v PVA, 0.006% w/v BSA, 2.4mM penicillin (Pen) and 16U/mL P'nase. The minimum detectable concentration was 1.7μM. The linear concentration range obtained for the BSA-PVA film was 7.5-283μM. The BSA-PVA P'nase biosensor detected penicillin in amoxycillin with an average percentage recovery of 97±12%. Higher penicillin concentrations (10-20ppm) were detected more successfully than lower concentrations (≤5ppm). These results indicate that further work is required to enable the successful detection of lower penicillin concentrations such as 5ppm.