The present study describes a novel and very sensitive electrochemical assay for determination of hydrogen peroxide (H2O2) based on synergistic effects of reduced graphene oxide‐ magnetic iron oxide nanocomposite (rGO‐Fe3O4) and celestine blue (CB) for electrochemical reduction of H2O2. rGO‐Fe3O4 nanocomposite was synthesized and characterized by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), X‐ray diffraction (XRD), electrochemical impedance spectroscopy and cyclic voltammetry. Chitosan (Chit) was used for immobilization of amino‐terminated single‐stranded DNA (ss‐DNA) molecules via a glutaraldehyde (GA) to the surface of rGO‐Fe3O4. The MTT (3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) results confirmed the biocompatibility of nanocomposite. Experimental parameters affecting the ss‐DNA molecules immobilization were optimized. Finally, by accumulation of the CB on the surface of the rGO‐Fe3O4‐Chit/ssDNA, very sensitive amperometric H2O2 sensor was fabricated. The electrocatalytic activity of the rGO‐Fe3O4‐Chit/DNA‐CB electrode toward H2O2 reduction was found to be very efficient, yielding very low detection limit (DL) of 42 nM and a sensitivity of 8.51 μA/μM. Result shows that complex matrices of the human serum samples did not interfere with the fabricated sensor. The developed sensor provided significant advantages in terms of low detection limit, high stability and good reproducibility for detection of H2O2 in comparison with recently reported electrochemical H2O2 sensors.