To improve the solubility and antitumor activity of ampelopsin, ampelopsin-loaded nanomicelles from the mixture of pluronic F127 and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS1000) were prepared by film-thin hydration method, in order to optimize the process conditions and physicochemical properties. The antitumor activities against MCF-7 cells between ampelopsin and nanomicelles were compared by MTT method, respectively. The results showed that the optimal nanomicelles were round with the nanometric size of (22.6±0.5) nm, encapsulation efficiency rate of (80.42±1.13)%, and drug-loading rate of (4.41±0.26)%. The solubility of ampelopsin in mixed nanomicelles significantly increased by 16 times. In different release media, the mixed nanomicelles could release more than 90% of drug in 8 h, and showed stronger cytotoxicity and inhibition against MCF-7 cells (P<0.01). The mixed nanomicelles can be used as new drug delivery system of ampelopsin.