A novel electrochemical sensor has been facilely fabricated by applying composite elements consisting of nitrogen and sulfur co-doped activated graphene (N,S-AGR) and molecularly imprinted polymer (MIP). N,S-AGR was synthesized by one-pot pyrogenation of a mixture of thiourea, KOH and graphene oxide, which was introduced to improve electron transfer capability and surface area of the electrode, while the electro-polymerized MIP layer afforded simultaneous recognition and quantification of cyclophosphamide (CPA) by utilizing Fe(CN)₆^3−/4− as probe to indicate electrical signals. Under the optimal conditions, a calibration curve of current shift versus concentration of CPA was got in the range of 8×10⁻¹²–8×10⁻⁷ mol L⁻¹, and the developed sensor gave a remarkably low detection limit (LOD) of 3.4×10⁻¹² mol L⁻¹ (S/N=3). Moreover, the as-prepared sensor illustrated other good merits like stability and selectivity, and played a role in real-time therapeutic drug monitoring after CPA administration in rabbit.