In this study, we have developed a self-standing nanocomposite mat, which combined of copper (benzene 1,3,5-tricarboxylic acid) MOFs (Cu(BTC)MOFs) and carbon nanofibers (CNFs) and used as an enzyme-free electrochemical sensor for detection of 4-nitrophenol in phosphate buffer (pH = 7). The high specific surface area and variety of functional active adsorption sites of as-developed hybrid mat facilitated faster transport of analyte molecules across the electrode-electrolyte interface. The Cu(BTC)MOF@CNF-modified glassy carbon electrode (Cu(BTC)MOF@CNF/GCE) demonstrated improved electrocatalytic responses toward 4-nitrophenol. The as-developed electrode showed that an excellent sensitivity (2.3429 μA μM−1 cm−2) with good selectivity and outstanding cyclic stability. The hybrid sensor exhibited a widespread linear range from 5.0 to 400 µM along with a low detection limit of 87.12 nM, for 4-nitrophenol determination. The present sensor electrode showed anti-interference property and high selectivity with good storage stability up to 30 days. Lastly, the sensing performances of the Cu(BTC)MOF@CNF/GCE toward para-nitrophenol in real samples give the satisfactory results, which proved the practical applicability of the present hybrid material. Furthermore, the real sample analyses results validated through HPLC results, and it was observed that the current results are in comparable with high-performance liquid chromatography data.