Here, a novel enzymatic biosensor was developed using multiwalled carbon nanotube including screen printed electrodes (MWCNT-SPE). Pyranose oxidase (PyOx) was immobilized on the electrode surface by way of gelatin membrane and then cross-linked using glutaraldehyde. Glucose was detected at -0.7 V (vs. Ag/AgCl) by watching consumed oxygen in enzymatic reaction after addition substrate. After optimization of pH and enzyme loading, the linearity was found in the range of 0.1–1.0 mM of glucose. After that, the effect of MCNT on the current was tested. Also the enzymatic biosensor including glucose oxidase instead of pyranose oxidase was prepared and the biosensor response followed for glucose. Furthermore, this system was tested for glucose analysis in soft drinks.