Non-small cell lung cancer (NSCLC) is a disease with high morbidity and mortality worldwide. NSCLC has significant metastasis ability, which is reported to be promoted by the process of epithelial-mesenchymal transition (EMT). The total flavonoid aglycone extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce cell apoptosis. Here we reconstructed TFAE (reTFAE) according to the main active components and revealed new mechanism in inhibiting EMT process of lung cancer. We provided the solid evidence that the compound mainly composed of baicalein, wogonin, and oroxylin-A presented inhibitory effect on the EMT process of A549 cells, and established the relationship between reTFAE, PI3K/Akt signaling pathways, TWIST1, and glycolysis pathway. The new mechanism we revealed and the new combination of research technologies including bioinformatics technology, chemical biology and molecular biology in our manuscript may be of tremendous interest to a broad range of academic, clinic and industrial researchers.