Thiosemicarbazone (TSC) matrices are constructed by electrochemically oligomerization on the graphite electrodes to investigate their matrix properties in terms of biomolecule immobilization for the biosensor applications. Amino functionalized oligomeric TSCs were successfully applied for the glucose oxidase (GOx) immobilization using glutaraldehyde (GA) as the crosslinker. Scanning electron microscopy (SEM) and fourier transform infrared spectroscopy (FT-IR) measurements are used to characterize the modified surface. Electrochemical measurements of the GOx biosensor were performed at ambient conditions at 0.7vs. Ag/AgCl electrode by following the oxygen consumption resulting from the enzyme activity, in the presence of glucose substrate. Linearity, operational stabilities and repeatability are investigated in detail, as well as optimization of pH and enzyme amount,. The optimized biosensor shows a very good linearity between 0.05mM and 1.0mM glucose with a 10s response time. Kinetic parameters such as K-m and I-max are also calculated. Besides the batch configuration, the analytical characteristics of the system were also evaluated in flow injection analysis (FIA) mode. Finally, the new biosensor configuration was applied for glucose determination on a real sample.