In this research, we have designed a phosphorous-doped tungsten trioxide nanosheet (P-WO 3 NS) for highly sensitive electrochemical sensors using the probe ultrasonication method. The synthesized P-WO 3 NS was characterized by a variety of instruments to understand their physicochemical and morphological properties. Subsequently, a glassy carbon electrode (GCE) modified with P-WO 3 NS/GCE was developed to investigate a highly sensitive detection of serotonin (ST) via electrochemical methods. The interaction between heteroatom P and WO 3 NS greatly increases the active sites, electrical conductivity properties, and electrocatalytic activity. The synergistic effect of the modified P-WO 3 NS/GCE exhibited excellent electrochemical performance, excellent catalytic activity, anti-fouling effect, high selectivity, and reproducibility for repeated ST detection. Also, ST is detected by linear sweep voltammetry with a very low detection limit of 60 nM (S/N = 3), good sensitivity of 0.2 μA μM −1 cm −2 , and a wide linear dynamic range from 10 nM to 100 μM. Furthermore, the practicability of the sensor was assessed in ST detection in spiked biological fluid samples, and good recovery results were achieved.