The influence of laser-conditioning on defects of SiO2 mono-layer films, which is laser conditioned by small spot laser scanning process, is investigated by the spot-size effect of laser-induced damage. The threshold and the density of defect were introduced to characterize the initiating defects in the film. It is found that the laser-induced damage threshold can be increased by a factor of 1.6 under the largest spot-size used and the defects with low threshold can be removed by laser conditioning scanning process.