The mutation of B-RafV600E is widespread in a variety of human cancers. Its inhibitors vemurafenib and dabrafenib have been launched as drugs for treating unresectable melanoma, demonstrating that B-RafV600E is an ideal drug target. This study focused on developing novel B-RafV600E inhibitors as drug leads against various cancers with B-RafV600E mutation. Using molecular modeling approaches, 200 blockbuster drugs were spliced to generate 283 fragments followed by molecular docking to identify potent fragments. Molecular structures of potential inhibitors of B-RafV600E were then obtained by fragment reassembly followed by docking to predict the bioactivity of the reassembled molecules. The structures with high predicted bioactivity were synthesized, followed by in vitro study to identify potent B-RafV600E inhibitors. A highly potent fragment binding to the hinge area of B-RafV600E was identified via a docking-based structural splicing approach. Using the fragment, 14 novel structures were designed by structural reassembly, two of which were predicted to be as strong as marketed B-RafV600E inhibitors. Biological evaluation revealed that compound 1m is a potent B-RafV600E inhibitor with an IC50 value of 0.05 μmol/L, which was lower than that of vemurafenib (0.13 μmol/L). Moreover, the selectivity of 1m against B-RafWT was enhanced compared with vemurafenib. In addition, 1m exhibits desirable solubility, bioavailability and metabolic stability in in vitro assays. Thus, a highly potent and selective B-RafV600E inhibitor was designed via a docking-based structural splicing and reassembly strategy and was validated by medicinal synthesis and biological evaluation.