Thymus quinquecostatus Celak., a traditional aromatic edible plant from Lamiaceae, is widely used as food additive, food condiment, spice, and herbal teas. Polyphenol-rich fraction of T. quinquecostatus (PRF) has been proven to be effective protective effect for cerebral ischemia reperfusion injury (CIRI) in our previous study. In this study, we developed a novel "Gut flora-Compound-Target-Pathway" (GCTP) network based on network pharmacology coupled with gastrointestinal metabolism for screening bio-active components, key targets and gut floras through the classical technique for order preference by similarity to ideal solution (TOPSIS). This compensates for the lack of gut floras and gastrointestinal metabolism in network pharmacology. Firstly, four incubation models covering simulated gastric juice, simulated intestinal juice, gut floras of normal and transient middle cerebral artery occlusion (tMCAO) rat in vitro were applied to PRF. The 109 proto-components and 64 metabolites were elucidated by ultra-high performance liquid chromatography-Q exactive orbitrap-mass spectrometry (UPLC-QE-Orbitrap-MS). Then, the key targets of matrix metalloproteinase 9 (MMP9), prostaglandin-endoperoxide synthase 2 (PTGS2), tyrosine-protein kinase fyn (FYN), estrogen receptor 1 (ESR1), amyloid precursor protein (APP), and epidermal growth factor receptor (EGFR), and gut floras of Enterococcus avium LY1 were selected. Moreover, the selected key proto-components were rosmarinic acid, daidzein, quercetin, luteolin, apigenin, methyl rosmarinate, kaempferol, luteoloside, and caffeic acid, and the key metabolites were isokaempferide, isorhamnetin, isoquercetin, and mangiferin. Binding of compounds to the key proteins was analyzed by molecular docking, and also verified though an 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) induced oxidative stress zebrafish model and real-time quantitative polymerase chain reaction (RT-qPCR) assays. This study provides a new idea and a better understanding of PRF for its protective effects on CIRI and its underlying mechanisms.