Nowadays, transition metal oxide (TMO), the semi-conductor molybdenum trioxide (MoO3) is under investigation. MoO3 is a good choice because of its many industrial applications and alluring qualities. It has a significant role as an industrial catalyst and has electrochromic properties. The first part is an experimental study of the optical behaviour of the above mentioned thin film onto various substrates. X-ray diffraction (XRD) analysis revealed an orthorhombic structure. Measurements were obtained of the average crystallite size and dislocation density, which were 132.8 nm and 5.7* 10− 4 nm-2, respectively. With the use of spectrophotometric measurements of transmittance (T) and reflectance (R) at normal incidence in the 300–1400 nm wavelength range at 310 nm thickness, the optical behaviour of the mentioned film onto various substrates was examined. The dispersion and oscillator energies were measured using the single oscillator model employing a Wemple–DiDomenico relation and indirect optical transitions with corresponding h ע energy gaps were identified for glass and quartz substrates. The second part introduces estimation optical behaviour of the above mentioned thin film using ANN model. Experimental data is used as inputs. The ANN modelling outputs optical characterization provide excellent results. Error values support the success of the modelling process (MSE > 10− 1). Moreover, the theoretical equation describing the experimental results was obtained depending on the ANN model which represents the relation between the inputs and outputs. According to this research, the ANN model can be utilized as an efficient tool to simulate and predict the optical parameters of the above mentioned film. Additionally, it can establish strong connections between theoretical and experimental fields.