The Cu-MOF@PANI@Ab modified SPCE used as a sensing platform. Cu-MOF and their composite were characterized by field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy (FTIR), RAMAN spectroscopy, X-ray diffraction (XRD), cyclic voltammetry (CV), and differential pulse voltammetry (DPV). The immunosensor possessed a dynamic linear range (10–105 pM) with a low limit of detection (LOD) of 0.33 pM (0.25 pg/ml) for CV and 0.17 pM (0.13 pg/ml) for DPV. The evaluation study of immunosensor displayed a good analytical recovery of ≥ 90%, when different concentration of thyroxine was added in the serum sample. In addition, to determine the reproducibility for working electrode between and within batch, the relative standard deviation calculated were 3.87% and 6.04%, respectively, represent it's good reproducibility. The immunosensor retained 90% of its initial activity after its regular use for 40 days, while being stored at 4 °C.