The invention relates to a glutathione-sensitive amphiphilic polyethylene glycol-hydroxycamptothecine conjugate, and further relates to synthesis of the conjugate, representation of the auto-agglutination property and the micelle size and hydroxycamptothecine releasing of the conjugate under the condition of a 37-DEG C phosphoric acid buffer solution. The structure of the conjugate is MPEG-X-COO-HCPT or HCPT-COO-X-PEG-X-COO-HCPT, wherein X is a linking group containing a disulfide bond, and COO is used for connecting the linking bridge X with HCPT through an ester bond. An in-vitro toxicity test and a tumor cell inhibitory activity test are conducted on human umbilical vein endothelial cells (HUVEC), human promyelocytic leukemia cells (K562), human colon cancer cells (HT-29) and human hepatocarcinoma cells (HepG2) by means of the synthesized compound separately, and results show that the compound has the significant anti-tumor activity, is low in toxicity to the normal cells and has a wide prospect on anti-tumor drug development and application.