A PEGylated 10-hydroxycamptothecin (HCPT) conjugate, an amphiphilic prodrug, in which two hydrophobic HCPT molecules were conjugated to the two ends of a hydrophilic poly(ethylene glycol) bis(carboxymethyl) ether (PEG-biCOOH) molecule, was synthesized by esterification of the terminal carboxylic groups of PEG-biCOOH and the 10-OH groups of HCPT molecules. The obtained conjugate, denoted as PEG-bi(COO-HCPT), was characterized using NMR, MALDI-TOF MS, HPLC, elemental analyses, FT-IR, and UV-Vis spectroscopy. Especially, the surface activity and self-assembly behavior of the amphiphilic conjugate in water were examined using equilibrium surface tension and fluorescence techniques. The prodrug exhibits a high drug content of similar to 52.7 wt% and the significantly enhanced water-solubility of HCPT from similar to 1.41 mu mol/L to similar to 2.04 mmol/L. The PEG-bi(COO-HCPT) in water can self-assemble into vesicles with similar to 130 nm in size at a concentration higher than similar to 23 mu mol/L, as evidenced using negative-staining, freeze-fracture, and cryogenic TEM, AFM, conductivity, fluorescence, and dynamic light scattering techniques. Furthermore, the cytotoxicity of the prodrug was evaluated against A549 non-small cell lung cancer (NSCLC) cells (Sensitive cells), human hepatoma (HepG2) cells (Non-sensitive cells), human umbilical vein endothelial cells (HUVEC, Normal cells), showing excellently improved cytotoxicity in comparison with pristine HCPT. This work provides a better understanding of self-assembly behavior of amphiphilic prodrugs in solutions and demonstrates that PEGylation of HCPT might be one of the promising strategies to improve its therapeutic efficacy.