In this study, a novel microbial biosensor was developed for the selective determination of L-Ascorbic acid. In the construction of the microbial biosensor, lyophilized Candida tropicalis yeast cells were immobilized with o-aminophenol by forming a film layer on a platinum electrode surface using electropolymerization. L-Ascorbic acid was quantified on the basis of both amperometric and differential pulse voltammetry (DPV) methods using the biosensor. The measurements were made at +0.24 V (vs Ag/AgCl) for amperometric studies and between 0.0 V and +0.7 V for DPV studies based on the oxidation of L-Ascorbic acid to dehydro-L-Ascorbic acid by ascorbate oxidase which takes place within the catabolic metabolic pathway of C tropicalis yeast cells. According to the results obtained from the two methods, the response of the biosensor depends linearly on L-Ascorbic acid concentration between 100 and 1500 I.LM. The detection limit was 62 mu M and 59 mu M for amperometric and DPV measurements, respectively. The response time of the microbial biosensor was 14 s and 5 s for DPV and amperometric measurements, respectively. In the optimization studies of the biosensor, some parameters such as the optimum amount of the microorganism, o-aminophenol concentration, pH and temperature were determined. For the characterization of the biosensor, reproducibility, storage stability and the effect of interferences were determined. (C) 2019 Elsevier B.V. All rights reserved.