In this work, pulsed potentiostatic reduction method was presented to directly construct a three dimensional graphene-nitrogen doped carbon nanotubes (3DG-NCNTs) network on the surface of a glassy carbon electrode (GCE). Scanning electron microscopy (SEM), Raman spectra and electrochemical experiments revealed the characteristics of the 3DG-NCNTs network. Further, such 3DG-NCNTs network was employed to immobilize a model molecule, hemoglobin (Hb), for the construction of an electrochemical nitromethane (CH3NO2) biosensor. The electrochemical properties of the biosensor were studied in detail, including the direct electron transfer of Hb and the electrochemical determination of CH3NO2. Due to the 3DG-NCNTs network, electrochemical biosensor demonstrated fast electron transfer rate (7.12 s(-1)) and excellent catalytic activity toward CH3NO2. Under optimal conditions, the proposed biosensor exhibited a wide linear response in the range of 5.0 x 10(-7)-3.5 x 10(-4) mol L-1, with a low detection limit of 1.5 x 10(-7) mol L-1. In addition, the biosensor had high reproducibility, stability and selectivity, which provided the possibility for the monitoring of CH3NO2 in real samples.