Rhubarb anthraquinone glycosides (RAGs) have been proven to have significant therapeutic effects on ischemic stroke, and this effect may be related to the microbiome-gut-brain axis. In this study, an HPLC-FLD method was established to measure brain-gut neurotransmitters of rats with cerebral ischemia-reperfusion injury (CIRI), to explore whether the mechanism of RAGs against CIRI is related to the microbiome-gut-brain axis. A Shimadzu ODS-3 C-18 column was used for chromatographic separation, and 5-hydroxytryptamine (5-HT), 5-hydroxy indole acetic acid (5-HIAA), glutamic acid (Glu), aspartic acid (Asp), and gamma-aminobutyric acid (GABA) were determined simultaneously. The results showed that there is an excellent linear relationship (R-2 >= 0.9990) and a high separation degree in the HPLC-FLD method. Whereas the contents of Asp and Glu in the brain and colon increased (p < 0.05), the contents of 5-HT, 5-HIAA, and GABA in the brain and colon decreased (p < 0.05) after CIRI. RAGs could effectively reduce the contents of Asp and Glu (p < 0.05), and increase the contents of 5-HT, 5-HIAA, and GABA in the brain and colon (p < 0.05). Combined with the previous experimental results, we can speculate that RAGs can regulate intestinal flora disorder caused by CIRI, and then regulate the imbalance between the release and decomposition of neurotransmitters caused by intestinal flora disorder.