Studies on the third-generation of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) targeting EGFR mutant remain hotspots, specifically for non-small cell lung cancer (NSCLC). In the current study, a new series of EGFR-TKIs with thieno[3,2-d]pyrimidine derivatives(6a-6r) bearing quinolin-2(1H)-ones were designed and synthesized, through conformational constrained strategy from the third generation of EGFR-TKI olmutinib. In vitro structure-activity relationship (SAR) studies indicated that compounds 6a, 6l, 6m, 6n and 6o exhibited good selective inhibition to EGFR (IC ≤ 250 nM) over wild type EGFR (IC > 10000 nM). The observed selectivity of compounds 6l and 6o was also proved by the computational molecular docking and the cellular thermal shift assay. These compounds had good growth inhibitory effect on the four tested cancer cell lines. Specifically, 6o could significantly inhibit the colony formation, wound healing and the expression of p-EGFR and its downstream p-ERK in EGFR H1975 lung cancer cells. Our findings suggest that the thieno[3,2-d]pyrimidine compounds, especially 6l and 6o, can selectively target the mutant EGFR in vitro and at cellular level and may serve as the lead compounds for generating new series of the third-generation EGFR-TKIs.