Abstract: Objective: To prepare hydroxycamptothecin (HCPT) loaded nanosphere made from poly(ethylene glycol)-poly(γ-benzyl-L-glutamate (PEG-PBLG) and study the drug release characteristic and pharmacokinetic of HCPT/PEG-PBLG nanosphere. Methods: HCPT/PEG-PBLG nanosphere was prepared using super diafiltration method; Scan electron microscope (SEM) was used to observe the shape of nanosphere; The HCPT release characteristic in vitro was evaluated by ultraviolet spectrophotometry. The high-performance liquid chromatography (HPLC) was used to detect and compare the pharmacokinetic parameters of HCPT/PEG-PBLG nanosphere and HCPT in rabbit plasma. Results: The shape of HCPT/PEG-PBLG nanosphere was core-shell structure and the nanosphere's size was about 230 nm, with 200 nm in core diameter and 30 nm in shell thickness. The drug loading capacity and drug encapsulation of HCPT/PEG-PBLG nanosphere were 7.5% and 56.8% respectively. The process of HCPT release from nanosphere in vitro and in vivo was composed of two steps, including abrupt-release and sustained-release. The main pharmacokinetic parameters of HCPT and HCPT/PEGPBLG nanosphere were as follows: the terminal elimination half-life (t_1/2β ) was 4.5 and 10.1 h, the peak concentration (C_max) was 2627.8 and 1513.5μ g ·L^-1, the apparent volumn of distribution (V_d) was 7.3 and 20.0 L and the area under the curve (AUC) was 2459.0 and 2175.9 μ g·h·L^-1. Conclusion: HCPT/PEG-PBLG nanosphere has the characteristics such as sustained-release and long-circulation, and can increased the affinity of HCPT to the tissues.