An enzyme immobilization technique has been developed to determine the concentration of biological compounds. This technique has been applied to penicillinase and urease, which are crosslinked as very fine films directly onto the sensitive ends of pH glass electrodes, thereby dispensing with the need of an on-line enzyme reactor. The biosensor is incorporated in an FIA system within a magnetically stirred detection cell. Penicillin-V in fermentation broth and urea in human serum samples were detected and the results were compared with HPLC and spectrophotometric methods. On-line measurement is achieved through the automation of this FIA system.