Tanshinone IIA (TA) is a major active component of Danshen (Salvia miltiorrhiza bunge), a well-known traditional Chinese medicine. In this paper, we describe an optimal method for preparing TA nanoemulsions (TA-NEs) to solve the problems of TA's poor solubility in water and insufficient dissolution rate. Optimization of the method for preparing nanoemulsions of lipid soluble TA by emulsification/high pressure homogenization was carried out with a 2(3) factorial design, using Tween 80, lecithin and water content as independent variables. The particle size, entrapment efficiency and polydispersity index of eight formulations were then evaluated. Two optimal formulations, TA-NE-F2 and TA-NE-F4, were developed and both exhibited markedly enhanced in vitro release ability, as compared to TA. However, only TA-NE-F4 remained stable for over 3 months. TA-NE-F4 particles were spherical and intact, with a mean particle size of 95.6 nm and a high entrapment efficiency of 99.3%, TA-NE-F4 showed a fast dissolution rate of 80% in 10 min and 100% in 20 min. The cytotoxicity of TA-NE-F4 against T24 human bladder cancer cells was 103.4-fold greater than TA alone in both a time- and a dose-dependent manner. Overall, we describe a feasible method for preparing TA-NEs that exhibit potent cytotoxicity.