A new and ultrasensitive impedimetric biosensor fabricated by using conjugated di-succinimide substituted polythiophene (P(ThidiSuc)) polymer modified indium tin oxide electrode is developed for the first time to detect the prostate specific membrane antigen (PSMA). The polymer P(Thi-diSuc) is synthesized by using a simple way and used in the fabrication of the proposed biosensor. The synthesized polymer contains di-succinimide groups, which offers covalent immobilization of PSMA specific antibodies. The developed strategy shortens the biosensor fabrication steps, because these active groups bind covalently to the amino ends of PSMA specific antibodies and this reaction does not require any crosslinking agent. Various characterization studies like impedimetric and voltammetric measurements, and morphological analyses are utilized to confirm the successful development of the biosensor. Under optimum conditions, the biosensing ability of the PSMA determination has a wide linear determination range from 0.015 to 14.4 pg mL(-1), as well as a low limit of detection of 6.4 fg mL(-1) and a high sensitivity of 1.36 kohm pg(-1) mL cm(-2). Furthermore, the proposed biosensor is able to measure the PSMA antigen in real human serums, which offers that it is a simple, low-cost, and sensitive tool with excellent potential for application in the quantification of PSMA.