SiO2 sol-gel antireflection film coated on fused silica can reduce the reflection loss and improve the transmittance of the optical component, although it is still prone to laser induced damage. Laser conditioning is an effective way to improve the laser induced damage threshold (LIDT) of SiO2 sol-gel antireflection film. In this paper, single-layer SiO2 sol-gel antireflection films pretreated by triple-frequency laser with different parameters are characterized by the macroscopical parameters, such as transmittance, refractive index, and thickness. The law of surface modification and the defect removal mechanism of the SiO2 sol-gel antireflection film by laser conditioning are obtained. It is found that laser conditioning can reduce the thickness of the film and introduce densification. In addition, laser conditioning can eliminate micro-defects, such as vacancies and voids in the preparation of SiO2 sol-gel antireflection films, which is the main reason to improve the laser damage resistance of films. Finally, the laser conditioning process with three step laser energy combinations of (0.2–0.6–1.0) Fth0 (zero damage threshold) is the best one to obtain high transmittance, and excellent effects on structure modification and defect removal of films. The research in this paper provides data support for the engineering application and mechanism research of laser conditioning.