Electrochemical methods have low toxicity, fast response and, easy operation. By modifying electrochemical sensors with a conductive and porous modifier, their sensitivity and selectivity can be improved. Nanomaterials with new and extraordinary properties are a new approach in science and especially in electrochemical sensors. In this study, UiO66-NH2/mesoporous carbon nitride (M − C3N4) composite provides a porous structure for decorated Au nanoparticles (AuNPs) to prepare a potent modifier for carbon paste electrode (CPE). Due to environmental toxicity of methotrexate, its sensitive, fast and, low-cost determination in workplace environments is of great interest. So, the modified CPE was applied as a sensitivity analysis approach for methotrexate in plasma samples. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used as techniques to optimize the analysis and measurement of methotrexate. To measure this drug, several effective parameters were optimized and a calibration curve was drawn under optimal conditions. The calibration curve showed a linear range from 0.5 to 150 μM with a detection limit of 0.15 μM for methotrexate. Examining the repeatability of the response of one electrode and multiple electrodes under optimal conditions shows the high precision of the developed method. Finally, this developed method based on UiO66-NH2/M-gC3N4/AuNPs|CPE was used to determine the methotrexate in the plasma sample using the standard addition method. Display omitted •UiO66-NH2/mesoporous carbon nitride composite provides decorated AuNPs.•A sensitive electrochemical sensor is used for analysis of methotrexate.•The ability of the developed method to measure methotrexate and acetaminophen in real plasma samples was investigated.