An amperometric biosensor based on zucchini (Cucurbita pepo) tissue homogenate is presented. The zucchini tissue homogenate was crosslinked with gelatine using glutaraldehyde and fixed on a pretreated teflon membrane. The zucchini tissue contained the enzyme ascorbate oxidase and this enzyme catalyzed the oxidation of ascorbic acid in the presence of dissolved oxygen. The principle of the measurements was based on the determination of the decrease in the dissolved oxygen level. Determinations were carried out by standard curves, which were obtained by the measurement of the decrease in the oxygen level related to ascorbic acid concentration. Optimization and characterization studies of the biosensor were carried out in detail. First of all, the amounts of zucchini tissue homogenate, gelatin, and glutaraldehyde percentage were optimized. Experimental parameters such as buffer system, pH, buffer concentration, and temperature were also optimized carefully. Thermal stability, storage stability, and repeatability of the biosensor were investigated. A linear response was observed from 5x10(-6) M to 1.2x10(-3) M ascorbic acid. Finally, the results of some plant and drug samples analyzed with the presented biosensor compared with the spectrophotometric method (Tillman reagent) used as a reference.