Biodegradable poly(epsilon-caprolactone)/poly(ethylene glycol) (PCL/PEG) copolymer nanoparticles showed potential application in drug delivery systems. In this article, monodisperse poly(epsilon-caprolactone)/poly(ethylene glycol)/poly(epsilon-caprolactone) (PCL/PEG/PCL, PCEC) nanoparticles, approximately 40 nm, were prepared by solvent extraction method using acetone as the organic solvent. These PCL/PEG/PCL nanoparticles did not induce hemolysis in vitro and did not show toxicity in vitro or in vivo. The prepared PCL/PEG/PCL nanoparticles were employed to load doxorubicin by a pH-induced self-assembly method. In vitro release study indicated that doxorubicin release from nanoparticles at pH 5.5 was faster than that at pH 7.0. The encapsulation of doxorubicin in PCL/PEG/PCL nanoparticles enhanced the cytotoxicity of doxorubicin on a C-26 cell line in vitro. Meanwhile, compared with free doxorubicin, doxorubicin in nanoparticles could more efficiently treat mice bearing subcutaneous C-26 tumors. The doxorubicin-loaded PCL/PEG/PCL nanoparticles might be a novel doxorubicin formulation for cancer therapy.