The invention provides environment-friendly road marking paint and a preparation method thereof. The paint comprises, by weight, 35-53 parts of water-based acrylic resin, 0.02-0.05 part of bactericide, 0.7-0.9 part of defoaming agent, 0.6-0.7 part of dispersing agent, 0.02-0.03 part of thickening dispersant, 1-2 parts of distilled water, 2-10 parts of titanium dioxide, 33-47 parts of inorganic filler, 0.01-0.02 part of wetting agent, 18-21 parts of glass beads, 4-5 parts of volatile solvent and 2-3 parts of film-forming agent. The preparation method comprises the following steps that the materials are prepared; the water-based acrylic resin and a part of the volatile solvent are evenly mixed in a dispersing machine; the bactericide, the defoaming agent, the dispersing agent, the thickening dispersant and the wetting agent are sequentially added and evenly mixed; the titanium dioxide powder, the inorganic filler and the glass beads are added in batches and uniformly mixed; the distilled water, the remaining volatile solvent and the film-forming agent are sequentially added; after all the raw materials are added, uniform stirring is conducted.