TNF-alpha, as a pro-inflammatory cytokine, regulates some physiological and pathological courses. TNF-alpha level increases in some important diseases such as cancer, arthritis, and diabetes. In addition, it displays an important function in Alzheimer's and cardiovascular diseases. Herein, a novel, sensitive, and selective voltammetric TNF-alpha immunosensor was prepared by using gold nanoparticles involved in thiol-functionalized multi-walled carbon nanotubes (AuNPs/S-MWCNTs) as sensor platform and bimetallic Ni/Cu-MOFs as sensor amplification. Firstly, the sensor platform was developed on glassy carbon electrode (GCE) surface by using mixture of thiol-functionalized MWCNTs (S-MWCNTs) and AuNPs. Then, capture TNF-alpha antibodies were conjugated to sensor platform by amino-gold affinity. After capture TNF-alpha antibodies' immobilization, a new-type voltammetric TNF-alpha immunosensor was developed by immune reaction between AuNPs/S-MWCNTs immobilized with primer TNF-alpha antibodies and bimetallic Ni/Cu-MOFs conjugated with seconder TNF-alpha antibodies. The prepared TNF-alpha immunosensor was characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), x-ray diffraction (XRD) method, x-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), thermogravimetric analysis, Fourier transform infrared spectroscopy (FTIR), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). A linearity range of 0.01-1.0 pg mL(-1) and a low detection limit of 2.00 fg mL(-1) were also obtained for analytical applications.