The efficacy and structural evolution of Mo-doped titania nanoparticles (MTNPs) as advanced photocatalysts for degrading methyl blue (MB) are investigated by X-ray absorption spectroscopy (XAS). The 3 wt % MTNP, characterized by uniform size and anatase structure, exhibits higher efficiency. The spectral analyses unveiled structural variations in the TiO6 octahedral structure and revealed an active site of the distorted square pyramidal structure symmetry (C4v). The in situ XAS spectra illustrate that MTNPs, particularly at 3 wt % doping, effectively enhanced the hole carriers in Ti 3d orbitals with a charge transfer to Mo 4d orbitals and impeded electron-hole pair merging, significantly enhancing the photodegradation under light illumination. This study deepens our understanding of the crucial role of Mo doping in optimizing TiO2 nanoparticle performance for efficient environmental remediation, showcasing the potential of MTNPs as sustainable photocatalytic materials.