The electrochemical behavior of nucleobases has been studied in 0.1 M phosphate buffer solution (PBS), pH 7.4 without removing oxygen, using glassy carbon electrode (GCE). Cyclic voltammetry (CV), electrochemical impedance experiment (EIS) and square wave voltammetry (SWV) were employed in the measurements of the nucleobases electrochemical signals at GCEs. Guanine (G) and adenine (A) produced well-defined oxidation peaks at about +0.66 and +0.96 V under the CV sweep at 100 mV/s, respectively. SWV was particularly useful in investigating the electrochemical behaviour of pyrimidine bases. In the SWV detection, thymine (T) clearly appeared an oxidation peak at 1.1 V, while cytosine (C) and uracil (U) did a complex oxidation peak at 1.3 V. Nucleobases possess an irreversible and adsorption-controlled electrochemical process at GCEs in 0.1 M PBS (pH 7.4). The average surface concentrations (Γ) of G and A on the surface of the GCE were estimated to be about 1.6515×10−10 and 8.8232×10−11 mol/cm2, respectively. Due to the selective interactions of nucleobases with each other, the nucleobase oxidation peaks shift and new oxidation peaks appeared. The new oxidation peaks at +1.62 and 1.55 V may correspond to the oxidation of GC pairs and AT pairs, respectively. The detection of the electrochemical behaviour and selective binding of nucleobases in a physiological PBS at pH 7.4 is of particular interest for electrochemical sensor applications in physiological media.