This study describes an orthogonal experimental design to optimize the formulation of cisplatin (CDDP)-loaded chitosan microspheres (namely, CDDP-DAC-MS) which were produced by an emulsion-chemical cross-linking technique. Seven factors and three levels for each factor that might affect the formulation of microspheres were selected and arranged in an L27(3(13)) orthogonal experimental table. A desirability function (df) calculated according to the trapping efficiency of CDDP, the drug content (%, w/w), and the size distribution of each batch of microspheres was introduced as an index of the microsphere formulation. The overall desirability functions (DF) were produced and treated by a statistic analytical system to optimize the formulation. Moreover, the contour maps were produced to analyze the influence of the seven factors on the size distribution, the drug content, and the drug trapping efficiency. The established optimum procedure was reproducible. Scanning electron micrographs showed that CDDP-DAC-MS were spherical with a coarse surface. The average diameter, drug content, and drug trapping efficiency of CDDP-DAC-MS were 74.8 microns, 20.8% (w/w), and 77.5%, respectively. The in vitro release of cisplatin from chitosan microspheres in saline was retarded compared with that from saline solution; the release of CDDP from chitosan microspheres was suggested to be controlled by the dissolution and diffusion of the drug from the chitosan matrix.