The aim of this study is to prepare cisplatin-incorporated nanoparticles based on ion complex formation between hyaluronic acid (HA) and cisplatin for antitumor drug delivery. To prepare nanoparticles using HA, bulk HA was degraded by hyaluronidases (HAses). Cisplatin-incorporated HA nanoparticles were prepared by mixing cisplatin with an aqueous solution of HA and then the nanoparticle solution was dialyzed to remove trace elements. Since glioma tumor cell lines are able to secrete HAse, extracts from U343MG and U87MG cell lines were used to test the release of cisplatin from the nanoparticles. The morphological observation of the cisplatin-incorporated nanoparticles showed that they had spherical shapes with a particle size around 100-200 nm. The loading efficiency of cisplatin in the nanoparticles was about 67-81% (w/w) and cisplatin was continuously released from the nanoparticles for 4 days. Especially, the release rate of cisplatin from the nanoparticles increased when HAse was added to the release medium. In the results of the HA zymography, the U343MG cell line secreted HAse, while the U87MG cell line did not. When the extracts from U343MG were added to the release medium, the release rate of cisplatin was slightly increased, while the extracts from U87MG did not significantly affect the release rate of cisplatin. In conclusion, cisplatin-incorporated nanoparticles have sufficiently small particle sizes to use as a drug targeting system. The release of cisplatin from the nanoparticles was responsive to the secretion of HAse. These nanoparticles are suitable vehicles for an antitumor drug targeting system.