The novel poly(cytosine)-modified glassy carbon electrode-based electrochemical sensor was fabricated potentiodynamically for the detection of Guanine (G) in clinical and biological samples. The surface of the electrode was successfully activated by electropolymerization, and about a 7.5-fold current improvement due to modification was achieved. From the analysis of the dependence of peak current and peak potential on a scan rate, a higher R 2 for the peak current on the square root of scan rate (R 2 = 0.999) than the dependence of peak current on scan rate (R 2 = 0.982) indicated that the oxidation of G at poly(cytosine)/ GCE was predominantly diffusion controlled. The oxidative peak response of the electrode revealed a high linear range of G concentration (0.1−200 μM) under optimized conditions. The detection limit and limit of quantification were 6.10 and 20.13 nM, respectively, associated with the %RSD of under 1%. The validation of the developed electrochemical sensor for the determination of G was investigated by analyzing human urine DNA and serum samples with spike recovery results in the range of 98.20−103.70% with the interferent recovery percentage in the range of 97.86−103.10% containing 50−300% of potential interferents. The newly designed sensor demonstrated the highest level of performance for the G detection in real samples.