An enzyme immobilization technique to measure penicillin concentration was developed. Penicillinase is immobilized by cross-linking with a very fine film of glutaraldehyde, which is sprayed on to the sensitive ends of two different pH glass electrodes. An extremely short response time of less than 10 s is guaranteed with electrodes made by this technique when measurements of penicillin are conducted in batch. The electrodes show good stability for 12 days when the sensor remains at room temperature. As the response time of the sensor is very short, an immediate determination of penicillin-V is possible by incorporating this enzyme electrode in a continuous-flow system, thereby dispensing with the need for an on-line enzyme reactor. The penicillin concentration is calculated as the difference in the potentiometric signal obtained as a peak when the sample flows past the sensor. Sample throughputs up to a maximum of 200 h−1 can be achieved. Samples are injected into a carrier stream consisting of a buffer of constant pH. Three different configurations of detection cells were designed and tested with the penicillinase electrode. First the performance of a magnetically stirred flow cell was studied. Subsequently a modified flow-through cell was tested, where the sensor was placed perpendicular to the direction of flow. A wall-jet cell was used to test the electrode where the flow direction coincided with the electrode axis. Finally, the magnetically stirred flow cell was used to detect penicillin-V in fermentation broth samples.