Abstract: To improve the physical property and bioactivity of methotrexate, this paper investigated the new formation of conjugate methotrexate-poly(ethylene glycol) and In vitro anti-tumor activity of the synthesized conjugate. The conjugate of methotrexate-poly(ethylene glycol), which was verified by the spectroscopy analysis of UV, IR and 13C NMR, was synthesized by chemical catalysis and micro-wave irritation. The determination for the conjugate of solubility in water and distribution coefficient in octanol-water system of the conjugate was done to examine its deliquescence property. The solubility in water and the distribution coefficient of the conjugate was greatly improved, which was increased by 128 folds and 5 folds, respectively. The In vitro anti-tumor activity of the conjugate was tested by mouse L1210 leukaemia cells, and the synthesized conjugate showed the same anti-tumor activity as the original methotrexate. Compared to the reported literature, the modification of methotrexate by poly(ethylene glycol) is more rapid and convenient.