The modified multifunctional electrodes for electro-Fenton (EF) process are suggested to be promising cathodes for in situ electro-generation and activation of H₂O₂ to produce hydroxyl radicals (^•OH). However, heterogeneous EF process still faces the challenges of limited catalytic activity and releasing of massive amounts of transition metals to the solution after removal of organic pollutants. The main aim of the present investigation was to prepare a cathode containing carbon nanotubes (CNTs) and CuFe nano-layered double hydroxide (NLDH) for degradation and mineralization of cefazolin antibiotic through electro-Fenton process. Structural and electrochemical analyses demonstrated that CuFeNLDH-CNTs nanocomposite was successfully incorporated on the surface of graphite cathode. Due to the increased formation of ^•OH in the reactor, the incorporation of CNTs into NLDH matrix with a catalyst loading of 0.1 g substantially improved the degradation efficiency of cefazolin (89.9%) in comparison with CNTs-coated (28.7%) and bare graphite cathode (22.8%) within 100 min. In the presence of 15 mM of ethanol, the degradation efficiency of cefazolin was remarkably decreased to 43.7% by the process, indicating the major role of ^•OH in the destruction of target molecules. Acidic conditions favored the degradation efficiency of cefazolin by the modified EF process. Mineralization efficiency of the bio-refractory compound was obtained to be 70.1% in terms of chemical oxygen demand (COD) analysis after 300 min. The gas chromatography-mass spectroscopy (GC-MS) analysis was also implemented to identify the intermediate byproducts generated during the degradation of cefazolin in the CuFeNLDH-CNTs/EF reactor.