The invention relates to a method for online coating and preparation of a diffusion polyester film. In terms of mass percentage, 70%-90% of a water-based organic polymer microsphere dispersion liquid having different microsphere particle sizes is slowly added to 9.9%-29.99% of pure water or deionized water and evenly stirred, then 0.01%-0.1% of a surfactant is added and evenly stirred, and a water-based coating liquid with the solid content being 17.5%-27% is prepared; polyester masterbatch containing 0-0.01% of a silica slipping agent is fused by an extruding machine, then a thick sheet is extruded, drawing is performed after casting, and one or two surfaces of a polyester film longitudinally drawn by 2.8-3.5 times are evenly coated with the prepared water-based coating liquid; the coated film is subjected to transverse drawing, drying solidification and heat setting and then is cooled, pulled and wound, and the diffusion polyester film with the coating thickness being 3-5 mu m is prepared. The film has high optical performance requirements and few appearance defects, procedures are simple and environment-friendly, and the production cost is low.