Acute kidney injury is among the most severe health problems today, with the greatest fatality ratios. The kidney injury molecule-1 (KIM-1) is considered to be a potential biomarker for diagnosis of the acute kidney injury. Herein, a sensitive, selective, and swift sandwich-type electrochemical KIM-1 immunosensor was fabricated based on porous NiCo2S4@CeO2 microspheres as a signal amplifier and covalent organic frameworks-gold nanoparticles (COFs-AuNPs) composite as an electrochemical sensor platform. The affinity of amino-gold between capture antibody and COFs-AuNPs composite led to immobilization of the capture antibody. The secondary antibody was then conjugated to NiCo2S4@CeO2 microspheres via electrostatic interactions. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), fourier-transform infrared spectroscopy (FTIR), x-ray diffraction (XRD) and x-ray photoelectron spectroscopy (XPS) techniques were performed to characterize the as-prepared materials. Some electrochemical characterization techniques including cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) were employed to gradually characterize the constructed immunosensor. The detection limit (LOD) of KIM-1 in plasma samples was calculated as 2.00 fg mL−1, making it an effective tool for the monitoring of acute kidney injury.