A novel, ultrasensitive impedimetric immunosensor was constructed for the detection of tumor necrosis factor alpha (TNF alpha) by using Poly(3-thiophene acetic acid) (P3), a conjugated polymer as an immobilization matrix. The polymer P3 contains a lot of carboxylic acid groups on its surface that provide a larger biorecognition surface. This developed immunosensor was prepared on hydroxy-bearing ITO surface via an ester bond linkage of polymer P3 to immobilize anti-TNF alpha antibodies. The ITO electrode modification steps and interaction between anti-TNF alpha antibody and TNF alpha antigen were monitored by cyclic voltammetry (CV) and by electrochemical impedance spectroscopy (EIS) method. After the analytical parameters optimization, a linear detection response from 0.01 pg/mL to 2 pg/mL, a limit of detection LOD of 3.7 fg/mL and a limit of quantification (LOQ) of 12.4 fg/mL were achieved, which provided accurate results (relative standard deviation; 4.03%). The characterization of this developed immunosensor was performed by using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), SEM-energy dispersive X-ray (EDX) mapping and atomic force microscopy (AFM). The immunosensor allowed a simple and fast detection of TNF alpha antigen in human serum and satisfied recoveries (98.69-105.20%) were obtained by using standard addition method.