A dual-functionalized electrochem. biosensor was developed for ultrasensitive detection of aflatoxin M1 (AFM1) in milk.AFM1, a hydroxylated derivative of aflatoxin B1 (AFB1), poses significant health risks, including reduced liver function, stunted growth in children, and immunosuppression.With no FDA-approved decontamination methods, a point-of-care (POC) device for rapid and precise AFM1 detection is crucial.The electrochem. biosensor combines specificity from COOH-functionalized AFM1-Aptamer (COOH-Apt) and o-hydroxyazobenzene polymers (Hazo-POPs) as a nanozyme probe at pencil graphite electrode (PGE).Hazo-POPs exhibiting integrated electroactive potential and peroxidase-like activity, independently utilized for AFM1 detection.Hazo-POPs were characterized using XRD, FTIR, UV-Vis, DLS, Raman spectroscopy, and FESEM and BET.At the same time, PGE surface modification was analyzed through optical microscopy, contact angle measurement, FESEM, micro-CT, CV, and EIS.In method, I, PGE was modified with Hazo-POPs@COOH-Apt for an optimized electrochem. response.COOH-Apt immobilization (Hazo-POPs@COOH-Apt/PGE) exhibited increased current, whereas AFM1 incubation (Hazo-POPs@COOH-Apt/PGE/AFM1) blocked the electron transfer, leading to decreased current.Method II employed Hazo-POPs' peroxidase-like activity, measuring the DPV response of 3,3',5,5'-tetramethylbenzidine (TMB), confirming aptananozyme (Hazo-POPs@COOH-Apt/PGE) specificity for AFM1.H2O2 generated hydroxyl radicals (°OH) in the presence of Hazo-POPs and TMB was oxidized to diimine by these radicals, leading to reduced TMB peak values, indicating oxidationThe electrochem. biosensor (Hazo-POPs@COOH-Apt/PGE) exhibited a broad linear range (0.005-500 nM) and low LOD (0.004 nM in method I; 0.003 nM in method II).Validated in spiked milk samples, it showed high recovery (101.21-104%), exceptional sensitivity, reproducibility, and stability for three weeks.