A simple one-step electrochemical deposition/activation of graphitic carbon nitride (g-C₃N₄) is highly desired for sensor configurations and remains a great challenge. Herein, we attempt an electrochemical route to exfoliate the g-C₃N₄ nanosheets in an aqueous solution of pH 7.0 for constructing a sensor, which is highly sensitive for the detection of serotonin (5-HT). The significance of our design is to exfoliate the g-C₃N₄ nanosheets, a strong electrocatalyst for 5-HT detection. Investigations regarding the effect of neutral pH (pH 7.0) on the bulk g-C₃N₄ and g-C₃N₄ nanosheets, physical characterization, and electrochemical studies were extensively carried out. We demonstrate that the g-C₃N₄ nanosheets have a significant electrocatalytic effect for the 5-HT detection in a dynamic linear range from 500 pM to 1000 nM (R² = 0.999). The limit of detection and sensitivity of the designed 5-HT sensor was calculated to be 150 pM and 1.03 µA µM⁻¹ cm⁻², respectively. The proposed sensor has great advantages such as high sensitivity, good selectivity, reproducibility, and stability. The constructed g-C₃N₄ nanosheets-based sensor platform opens new feasibilities for the determination of 5-HT even at the picomolar/nanomolar concentration range.