A self-assembly method was used to synthesize the Auricularia auriculajudae polysaccharides (AAPs)-cisplatin (cis-dichlorodiammine platinum (II), CDDP) complexes. Response surface methodology (RSM) was used to optimize the synthesis conditions. The effects of four independent factors: pH (X1: 1–9), time (X2: 0.5–3h), temperature (X3: 20–70°C), and amount of CDDP (X4: 2–22mg) on the platinum content in AAPs-CDDP complexes were evaluated. The RSM results showed that the optimal synthesis conditions were: X1: 7.7, X2: 1.83h, X3: 56.93°C, and X4: 6.02mg. Verification tests showed no significant differences between the practical and the predictive values for each response. Under the optimal conditions, platinum content was 47.0mg/g, without significant differences with the predicted value (47.1mg/g). The FT-IR analysis and 1H NMR spectra of AAPs-CDDP complexes showed that the AAPs combined with CDDP using the hydroxyl group on the polysaccharide’s chains. In addition, the microphotograph of the AAPs and AAPs-CDDP complexes suggested that an AAPs-CDDP complex was formed. The MTT assay in vitro indicated that AAPs-CDDP complexes exhibited significant anti-tumor activity against Hela and LoVo cells.