Hydroxycamptothecin (HCPT) is valid to various malignant tumors, but its insoluble and unstable lactone ring in physiological environment have restricted the clinic application. This work was aimed to formulate HCPT-loaded poly(DL-lactic acid)-poly(ethylene glycol) (PELA) fibrous mats through blend electrospinning with 2-hydroxypropyl-beta-cyclodextrin (HPCD) to modulate the drug release and matrix degradation, and to enhance the structural integrity and cytotoxicity of the released HCPT. The entire drug fraction retained its active lactone form within electrospun fibers, and that was maintained over 85% during incubation for over 1 month. A biphasic release pattern was determined for HCPT-loaded electrospun fibers, which can be modulated by the addition of HPCD. HPCD served as solubilizer to maintain a large concentration gradient for HCPT between saturation and diffusion, and liberated HPCD created microstructure of ultrafine fibers, leading a faster release profile in the second phase. In vitro cytotoxicity test showed over 7 times higher inhibitory activity against cancer cells for HCPT-loaded electrospun fibers than free drug during 72h incubation. Higher apoptosis rates and the arrest of the cell cycle during the S and G(2)/M phases were detected through flow cytometry analysis. It indicated therapeutic potentials of HCPT-loaded electrospun fibers as implantable anti-cancer agents for local chemotherapy.