We report on the electrochemical determination of one the most effective and widely used chemotherapeutic, anti-inflammatory, and immunomodulator agents, methotrexate (MTX), using low-cost, green, and facile one-pot prepared graphitic carbon nitride (g-CN ) nanosheets. The g-CN nanosheets have been characterized utilizing Fourier transform infrared spectroscopy, X-ray diffraction(XRD), scanning electron microscopy(SEM), and density functional theory (DFT). In comparison to the bare carbon paste electrode (CPE), the g-CN -modified electrode showed a spectacular enhancement in the electrochemical oxidation and detection abilities of MTX. The proposed material exhibits very low limits of detection (12.45 nM) and quantification (41.5 nM), while possessing a wide linear range of 0.22–1.11 μM and 1.11–27.03 μM under optimized conditions at pH 7.0. Due to the ease of preparation of g-CN, it can be adopted for the cost-effective detection of MTX in industrial and clinical analyses.