The importance of nanodiamond in biological and technological applications has been recognized, and applied in drug delivery, biochip, sensors and biosensors. Nanodiamond (ND) and nitrogen doped nanodiamond (NND) films were deposited on n-type silicon films, and later functionalized with enzyme glucose oxidase (GOX). Functionalized electrode has been characterized using different techniques; i.e.fourier transform spectroscopy (FTIR) -, Raman spectroscopy, atomic force microscopy (AFM) and electrochemical techniques, respectively. Under this work, the ND/GOX and NND/GOX electrodes have demonstrated providing sensitive glucose concentration response. Besides, the cytotoxic effects of the NDs have been studied in vitro. Human Embryonic Kidney 293 (HEK293) cells are cultured in the presence of the films then toxicity has been detected using MTT-based cytotoxicity assays utilizing 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT). The final results for MTT assays are quantified by spectrophotometry using a plate reader at 570 nm As-prepared nanodiamond has been found to be stable , biocompatible and useful for biosensing applications. A linear response of the enzyme based electrode to glucose concentration is also observed from 1-8 x mM before saturation condition close to 10mM has been observed.