A series of new [1,2,4]triazolo[4,3-a]pyrimidine derivatives was prepared using a one-pot three-component synthesis from 5-amino-1-phenyl-1H-1,2,4-triazoles, aromatic aldehydes and ethyl acetoacetate. The compound structures were confirmed by IR,1H-NMR,13C-NMR, HRMS and X-ray analyses. The biological activity of these compounds as antitumor agents was evaluated. Their antitumor activities against cancer cell lines (MDA-MB-231 and MCF-7) were tested by the MTT in vitro method. Among them, compounds4cand4jdisplayed the best antitumor activity with IC50values of 17.83 μM and 19.73 μM against MDA-MB-231 and MCF-7 cell lines, respectively, compared to the Cisplatin reference.