The present work focuses on the structure, morphology, optical and sensing analysis of Electron Beam Irradiation (EBI) treated WO3 films synthesized by the spray pyrolysis technique. X-ray diffraction (XRD) revealed a slight shift in the 2θ position, indicating stress generated due to EBI process. Scanning Electron Microscopy (SEM) morphographs presented well-defined grains at higher irradiation dosages. X-ray Photoelectron Spectroscopy (XPS) studies showed increased oxygen vacancy defects for the 5 kGy treated sample compared to unirradiated WO3. Sensing analysis of unirradiated and EBI-treated films was conducted towards ammonia (NH3) at an optimum operating temperature of 200 °C. Sensor response of 5 kGy treated film increased by − 4.5 fold compared to unirradiated film at 5 ppm NH3 concentration. The current study demonstrates the importance of EBI in tailoring the NH3 sensing properties of WO3 films.