LA–PEG–G molecule was prepared through the covalent conjugation of linoleic acid, polyethylene glycol and guanosine. LA–PEG–G molecule can self-assemble into spherical aggregate in aqueous solution. The size of the LA–PEG–G aggregate was approximately 132.1 ± 13.718 nm, and the Zeta potential was −36.3 ± 0.9644 mv. The loading rate of LA–PEG–G aggregates to Dox was 84.62 ± 1.810%. In vitro release experiments showed that Dox release from the LA–PEG–G–Dox nanomedicine was slow-release. Cell experiments showed that LA–PEG–G aggregates have low cytotoxicity, and the relative cancer cells survival rate decreased with the increasing of time and LA–PEG–G–Dox nanomedicine concentration. In vivo experiments showed that LA–PEG–G–Dox nanomedicine can inhibit tumor growth, and the LA–PEG–G aggregate could also delay the tumor growth which is very important to play the synergistic antitumor effect with the anticancer drug. Serum biochemistry showed that LA–PEG–G–Dox nanomedicine which reduced the Dox toxicity had very good antitumor effect. The tumor tissue sections indicated that LA–PEG–G–Dox nanomedicine can effectively induce tumor cell apoptosis, and finally cause tissue necrosis to achieve tumor treatment.