The invention discloses a preparation method of regenerated cellulose fiber-modified rhamnolipid-emulsified asphalt, and belongs to the technical field of asphalt preparation. The preparation method comprises the following steps: preparation of regenerated cellulose fibers, inoculation of pseudomonas aeruginosa in a slant culture medium, inoculation in a seed culture medium, plasma mutagenesis, inoculation in a plate culture medium, inoculation in a fermentation culture medium, acetic acid extraction, and preparation of emulsified asphalt. The regenerated cellulose fibers are used for modification to improve physical and mechanical properties of asphalt; rhamnolipid has relatively strong surface activity and emulsifying ability, and is completely biodegradable and environment-friendly; the asphalt prepared by the invention has good emulsifying effect, good stability, and excellent filtration reducing property and inhibition; plasma is used as a mutagen to mutagenize strains of pseudomonas aeruginosa, so that the yield of obtained rhamnolipid is higher; the regenerated cellulose fibers and other materials are pretreated by the plasma so as to enhance compatibility of the fibers with the asphalt and bonding strength of two-phase components.