In this paper, the interactions between interstitial oxygen atoms and substitutional atoms in Ti-Nb-O alloys were investigated by first-principles calculations based on the density functional theory. The binding energies and formation energies of oxygen atoms sited were calculated in various interstices with different geometries, element species, and atomic arrangements. The calculation results showed that there was a strong repulsive binding energy between the interstitial oxygen and the substitutional niobium atoms. On this basis, the energetically favored sites and the distribution of interstitial oxygen atoms in Ti-Nb-O alloys were discussed and demonstrated by an electron energy loss spectroscopy method. Furthermore, the diffusion activation energies of interstitial oxygen atoms based on the formation energy results were presented, and the diffusion behaviors of interstitial oxygen atoms in Ti-Nb-O alloys were also discussed.