A series of novel thieno[3,2-d]pyrimidine derivatives possessing diaryl semicarbazone scaffolds were designed, synthesized and evaluated for their anticancer activity. Most compounds displayed good to excellent potency against four tested cancer cell lines as compared with GDC-0941 and sorafenib. In this study, a promising compound 36 (PI3Kα IC50 = 0.027 μM) was identified, which showed the most potent antitumor activities with IC50 values of 0.057 μM, 0.039 μM, 0.25 μM, and 0.23 μM against H460, HT-29, MKN-45 and MDA-MB-231 cell lines, respectively. In addition, the SAR analyses indicated that compounds with 4-morpholino group at the C-4 position of thieno[3,2-d]pyrimidine moiety exhibited superior activities than compounds bearing chain amino groups. In addition, compounds with mono-methoxy group at the 3-position or dimethyl groups at the 3,5-position on the terminal phenyl ring were more active. The SAR analyses will guide us to further refine the structure of the thieno[3,2-d]pyrimidine derivatives to achieve optimum anticancer activity.