Well-crystallized vanadium pentoxide V2O5 thin films are fabricated on MgO single crystal substrates by using pulsed-laser deposition technique. The linear optical transmission spectra are measured and found to be in a wavelength range from 300 to 800 nm; the data are used to determine the linear refractive index of the V2O5 films. The value of linear refractive index decreases with increasing wavelength, and the relationship can be well explained by Wemple’s theory. The third-order nonlinear optical properties of the films are determined by a single beam z-scan method at a wavelength of 532 nm. The results show that the prepared V2O5 films exhibit a fast third-order nonlinear optical response with nonlinear absorption coefficient and nonlinear refractive index of 2.13 × 10−10 m/W and 2.07 × 10−15 cm2/kW, respectively. The real and imaginary parts of the nonlinear susceptibility are determined to be 3.03 × 10−11 esu and 1.12 × 10−11 esu, respectively. The enhancement of the nonlinear optical properties is discussed.