The invention discloses non-dissolving antibacterial cellulose acetate and a preparation method and application thereof; the non-dissolving antibacterial cellulose acetate is prepared by substituting hydrogen on part of hydroxyl groups in cellulose acetate by bromoacetyl groups. The preparation method comprises: 1) dissolving the cellulose acetate; 2) dropwise adding bromoacetyl under quick stirring to ensure the temperature of a reaction system is kept at 0-5 DEG C; 3) allowing reacting at 20-35 DEG C under quick stirring; 4) adding deionized water to precipitate out cellulose acetate, and drying to obtain the non-dissolving antibacterial cellulose acetate. An antibacterial reverse osmosis membrane is prepared by dissolving the non-dissolving antibacterial cellulose acetate in a solvent, adding a non-solvent to obtain a casting liquid, scraping, and solidifying. According to the technical scheme provided by the application, bromo-alkyl antibacterial groups are grafted to cellulose acetate to obtain the non-dissolving antibacterial cellulose acetate having good chemical stability and good antibacterial efficiency, and a reverse osmosis membrane made with the non-dissolving antibacterial cellulose acetate has good antibacterial property.