The invention discloses a preparation method of cherokee rose polysaccharide derivatives with an antitumor activity. The preparation method comprises the following steps: firstly smashing cherokee rose fruit, degreasing, removing oligosaccharide, lixiviating with hot water twice, then mixing extracts, filtrating, concentrating, precipitating and drying so as to obtain crude cherokee rose polysaccharide; adding diethylin ethyl amino ethyl (DEAE)-cellulose 52 to carry out static absorption for 30 minutes; washing with distilled water, filtering, and collecting filtrate so as to obtain the decolored crude cherokee rose polysaccharide; carrying out protein removal on a crude polysaccharide solution by using an enzyme method and a Sevag method; further separating the crude cherokee rose polysaccharide with the DEAE-cellulose 52, collecting, dialyzing, and freeze-drying so as to obtain cherokee rose polysaccharide; and finally, carrying out sulphating, carboxymethylation and hydrochloric acid degradation on the cherokee rose polysaccharide, and carrying out carboxymethylation on degradation products so as to obtain a plurality of cherokee rose polysaccharide derivatives. The plurality of cherokee rose polysaccharide derivatives have in vitro antitumor characteristics, and can be used for inhibiting the growth of three tumor cells, namely, ovarian cancer cells, liver cancer cells and rectal cancer cells.