Tryptanthrin has not been widely applied in clinical practice due to its poor solubility and low bioavailability in spite of possessing several biological and pharmacological activities. Here, to improve the solubility of tryptanthrin, two types of novel tryptanthrin-loaded micelles were prepared. One was tryptanthrin physically encapsulated by distearoyl phosphatidylethanolamine polyethylene glycol (DSPE-PEG) and the other was pegylated tryptanthrin synthesized by acid-sensitive hydrazone bond and further prepared as micelles. Molecular imprinting technology was used to separate pegylated tryptanthrin and free mPEG-COOH in the preparation of PEGylated tryptanthrin micelles with considerably high separation efficiency. The solubility of tryptanthrin-loaded DSPE-PEG micelles (TDMs) and PEGylated tryptanthrin micelles (PTMs) was increased by 300 and 1493 fold compared with that of tryptanthrin, respectively. The PTMs increased the solubility of tryptanthrin more effectively and 95% of tryptanthrin was released from PTMs at pH 5.5 in 12 h. The cytotoxicity of PTMs decreased under physiological conditions compared with that of tryptanthrin, whereas at pH 5.5, the PTMs showed comparable cytotoxicity with that of tryptanthrin, indicating successful drug release from the carrier in response to tumor cell pH. Overall, we elucidated an efficient method to improve water solubility of tryptanthrin and indicated pegylated tryptanthrin is a promising prodrug.