To develop biodegradable docetaxel-loaded self-assembled nanoparticles of poly (D,L-lactide-co-glycolide)/hyaluronic acid block copolymers were successfully synthesized. These copolymers could form nanoparticles with small size (<200 nm), an acceptable CMC (~7.9 mg/L), typical core/shell structure and superior stability in one week. DTX-loaded PLGA(502H)-b-HA(5.6k) nanoparticles (DTX/SANPs) showed a biphasic release pattern within 120 h, and exhibited enhanced cytotoxicity toward CD44-overexpressing MDA-MB-231 cells. Cellular uptake study indicated that PLGA(502H)-b-HA(5.6k) nanoparticles (SANPs) were taken up in MDA-MB-231 cells by CD44-mediated endocytosis. Pharmacokinetics study revealed DTX/SANPs could prolong the circulation of DTX in the blood. In vivo studies demonstrated that SANPs exhibited enhanced tumor targeting and antitumor activity with lower systemic toxicity. In conclusion, DTX/SANPs have great potential for targeted chemotherapy for CD44-overexpressing breast cancer.