Aflatoxin M (AFM), a hydroxylated metabolite of aflatoxin B (AFB), contaminates milk and dairy products from livestock ingestion of feed contaminated with a species of . As AFM is a Group-II B carcinogen, it is indispensable to develop novel, easy-to-handle, sensitive, portable and cost-effective strategies for its detection. Herein, a covalent organic framework (COF)-based electroactive nanocomposite, AgO/g-CN-COOH@MA-DBB-COF (silver oxide/carboxy-functionalized graphitic carbon nitride@melamine-dibromo butane COF), is designed to serve as a multifunctional receptor surface. The AgO/g-CN-COOH@MA-DBB-COF formation was characterized through X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, micro-Raman spectroscopy (RAMAN), dynamic light scattering (DLS) and thermogravimetric analysis (TGA), and each step of the sensor fabrication was monitored using field emission scanning electron microscopy (FE-SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Under the optimal conditions, the designed immunosensor permitted the detection of AFM in the linear range of 0.03-1000 fg mL, with a 0.01 fg mL limit of detection (LOD). The selectivity of the designed immunosensor was validated an anti-interference study. The practical applicability of the immunosensor was demonstrated by the detection of AFM in real milk samples, and good recovery values (97.28-102.62%) were obtained. Furthermore, the developed immunosensor and high-performance liquid chromatography (HPLC) were employed in parallel to detect AFM in local market milk samples from twenty different sites to validate the performance of the newly designed immunosensor. Additionally, the designed immunosensor was stable over an extended period of time. This work reports a single-step field-portable multifunctional innovative electrochemical immunoreceptor design for on-site and label-free detection of AFM in milk samples. Hence, the present study is the first report on the fabrication of a multifunctional innovative electrochemical immunoreceptor based on PGE/AgO/g-CN-COOH@MA-DBB-COF for the detection of AFM in milk samples.