Objective: To evaluate the antitumor activity of a novel class of 4,8-Disubstituted-8,9-dihydropyrazine[2,3-g]quinazoline-7(6H)-ketones in vitro,and to screen potential anticancer compounds for further study. Methods: Seventeen compounds of 4,8-Disubstituted-8,9-dihydropyrazine[2,3-g]quinazoline-7(6H)-ketones were synthesized with solid-phase method for biological evaluation of EGFR tyrosine kinase.MTT method was used to evaluate the cytotoxic activity in vitro against three human cancer cell lines (human lung carcinoma cell line A549,human leukemia cell lines K562 and human gastric carcinoma cell line SGC7901). Results: Compound 7-13 and 7-14 showed potent antitumor activities against A549 cells,with IC_(50) values of 8.10 and 8.12 mol/L, respectively.Eight compounds showed proliferative inhibition effect on K562 cells,especially 7-2,7-13 and 7-17,with IC_(50 )values of (2.22),0.57 and 7.20 mol/L,respectively.And compound 7-13 and 7-3 showed potent antitumor activity against SGC7901 cells,with IC_(50) values of 4.20 and 9.71 mol/L, respectively. Conclusion:The synthesized compounds 4,8-Disubstituted-8,9-dihydropyrazine[2,3-g] quinazoline-7(6H)-ketones show inhibition effects on human cancer cell lines in vitro. Compound 7-13 has anticancer activity in all three cancer cell lines, which might be used as a potential antitumor drug for further study.