In order to search for new antitumor drugs with high efficiency and low toxicity, a series of novel 4-aminoquinazoline derivatives containing 1,3,4-thiadiazole group were designed, synthesized and evaluated for antiproliferative activity against four human cancer cell lines (H1975, PC-3, MCF-7 and HGC-27) using MTT assay in vitro. Among them, compound N-(5-((3,5-dichlorobenzyl)thio)-1,3,4thiadiazol-2-yl)-2-((4-(phenylamino)quinazolin-2-yl)thio)acetamide ( 15o ) showed good anti-tumor proliferation activity against four tested cancer cell lines, with IC 50 values of 1.96±0.15μM to PC-3 cell. The antitumor activity was significantly better than that of gefitinib. Further mechanism studies showed that compound 15o inhibited the migration ability of PC-3 tumor cells in a concentration dependent and time-dependent manner, and blocked the cell cycle in S phase. At the same time, cell cloning experiment further proved that compound 15o significantly inhibited the colony formation of PC-3 cells, The inhibition rate was as high as 92% at 2.0 μM.