This paper presents the construction of various biosensors using thin-film layers incorporated in flow injection devices, providing automated systems for biomedical analysis, process and environmental monitoring. A urease sensor has been developed in conjunction with a flow injection system for the automatic determination of urea. Use of the spraying immobilization technique gives rise to a response time of a few seconds, which allows sample throughputs up to 200 h−1. With a penicillin biosensor adapted in an appropriate cell detection, on-line measurements of penicillin V in the fermentation broth are achieved during the whole fermentation process; the results are compared with the HPLC method. Linearity, sensitivity and reproducibility of the biosensor are studied with regards to sample dilution in a stirred flow detection cell to provide optimal operating conditions. Measurements without any change in parameters are obtained during the whole fermentation process. Acetylcholinesterase sensors have been used in batch systems for the determination of pesticides, but they require large amounts of substrate. When those enzyme sensors are combined with flow injection systems, only small volumes (100 μl) of substrate are injected into the carrier stream and an automated system can be obtained for continuous control of water quality.