The purpose of a drug delivery system is to efficiently deliver drugs to a desired target, while simultaneously reducing the side effects caused by these drugs and maximizing their efficacy. However, in the manufacture of a drug delivery system, it is difficult to control the amount of drug encapsulation. In this study, we developed a simple formation process of self-assembled hydrogels that made it easier to package the desired amount of anticancer drugs. A self-assembled hydrogel was prepared by simply mixing transferrin, dithiothreitol, and an anticancer drug in a salt solvent. The structural conditions of the hydrogel were determined in order to control the concentration of the transferrin protein, dithiothreitol, and salt in the solvent. The self-assembled hydrogels contained the desired amount of anticancer drugs. With this system, changes in pH and temperature control the release rate and the release ratio of anticancer drugs. The cytotoxicity of the drug-loaded hydrogel was evaluated, which showed that 80% of the treated cells had been killed following 48 h of incubation.