A novel impedimetric immunosensor based on conjugated succinimide group substituted polythiophene polymer (SucS-PThi) modified indium tin oxide (ITO) electrode was fabricated for ultra-sensitive and ultra-selective determination of kallikrein 4 (KLK 4) antigen in diluted human serum. The basic reason for the usage of SucS-PThi polymer was that its rich effective functional groups and biocompatibility. KLK 4, an important prostate biomarker was the target analyte and anti-KLK 4 antibodies immobilized electrode was the diagnostic tool for KLK 4 antigen detection. The fabricated biosensor was characterized by employing electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), scanning electron microscopy (SEM) and energy dispersive X-ray (EDX), fourier transform-infrared (FTIR) and Raman spectroscopy. Under optimized conditions, a linear concentration range from 0.04 pg/mL to 30 pg/mL and a low detection limit (LOD) of 12.2 fg/mL were obtained and the LOD was lower than of most of the existing techniques, illustrating its feasibility for practical application. Moreover, the developed sensing system combined the outstanding properties in terms of selectivity, sensitivity, repeatability, reproducibility and reusability, without requiring for labor - intensive labeling stages. In addition, it was successfully utilized for accurate quantification of KLK 4 antigen in human serum samples.