The work aims to investigate the effect of V2O5 film thickness on laser damage resistance. V2O5 thin films with different thickness were prepared on optical grade single crystal diamond substrates by radio frequency reactive magnetron sputtering. The optical response curves of the thin films were tested by a pulsed laser with a pulse width of 10 ns and a wavelength of 1064 nm. The changes of transmittance and the switching time before and after phase transformation were measured to determine whether the thin films were damaged, and the laser damage threshold was obtained according to the damage probability. The prepared films were polycrystalline V2O5 with single component and had obvious preferred orientation on (001) surface. At the same film thickness (350 nm), with the increase of laser energy density, the phase transition closure time of the films decreased monotonously from 1.48 ms to 0.64 ms, and the phase transition recovery time increased monotonously from 11.6 ms to 20.4 ms. The transmissivity decreased monotonously from 19% to 8%, and the transmissivity decreased monotonously from 77% to 51%. In the range of 150~550 nm of film thickness, the laser damage threshold increased first and then decreased with the increase of the film thickness; When the film thickness was 250 nm, the laser damage threshold reached the maximum value of 260 mJ/cm2; when the film thickness was 550 nm, the minimum value was 209 mJ/cm2. The thickness of V2O5 film has great influence on laser damage resistance. Reasonable control of film thickness can effectively improve the laser damage threshold, thus improving the laser damage resistance of V2O5 film based on diamond substrate.