Abstract Objectives Sensitive and accurate techniques for early detection of prostate cancer, which has a good chance for successful treatment if detected early, are of utmost value. Our aim is to develop a sensitive chronoimpedimetric biosensor for detection of circulating prostatic tumor cells (CTCs) with an aptamer selective for prostate specific membrane antigen (PSMA). Methods Thiolated PSMA-specific aptamer was immobilized on the gold nanoparticle modified carbon screen-printed electrodes. After characterization with cyclic voltammetry and electrochemical impedance spectrometry, scanning electron microscopy and atomic force microscopy studies were conducted to confirm the modifications. LNCaP cells (androgen-sensitive human prostate adenocarcinoma cells), were then added to the serum samples and chronoimpedimetric detection of CTCs in samples were performed. Results Our study showed one cell detection capability in real serum samples with a linear range from 1 to 40 cells/mL. The incubation time was 130 s. LOD was found to be 0.62 cells/mL and relative standard deviations were lower than 2% RSD. Reproducibility tests indicated a regression coefficient as R2 = 0.9963 ± 0.0178. Conclusions This new biosensor enables rapid, accurate, precise, reproducible and highly sensitive detection of PSMA on CTCs in prostate cancer and paves the way to new diagnostic applications and research-based studies.