This paper demonstrates a novel strategy for real-time monitoring of cellular small biomolecules based on graphene hybrid composites film, which was fabricated by one step electrodeposited reduced graphene oxide (ERGO) incorporating adsorption of electroactive methylene blue (MB) onto glassy carbon electrode (GCE). UV-visible spectroscopy, FT-IR spectroscopy, electrochemistry and scanning electron microscopy were employed for systematical characterization of GO-MB adsorptive nanostructure. The ERGO-MB/GCE shows more favorable electron transfer kinetics for potassium ferricyanide and potassium ferrocyanide probe molecules, which are important electroactive compounds, compared with bare GCE, MB/GCE and ERGO/GCE. The electrochemical behaviors of dissolved oxygen and hydrogen peroxide (H2O2) at ERGO-MB/GCE were investigated by cyclic voltammetry, suggesting that the modified electrode exhibits enhanced electrocatalytic activity toward H2O2. Under physiological condition, the ERGO-MB/GCE showed a linear amperometric response from 0.5 mu M to 11.68 mM for H2O2, with the detection limit of 60 nM (S/N = 3), and also showed high sensitivity (102 A mu M-1- cm(-2)) and short response time within 2 s for H2O2 detection. Besides, the reproducibility, stability and anti-interference ability of the proposed nonenzymatic H2O2 biosensor were also proved. The developed method has been successfully applied to determination of H2O2 released from living cells, which could be clearly distinguished cancer cells including Hep3B, HepG2 and HeLa cells from normal cells HEK 293. This assay shows great potential for nonenzymatic determination of H2O2 in physiological and pathological investigations. (C) 2016 Elsevier B.V. All rights reserved.