Based on network pharmacology, molecular docking and in vitro experiments, the mechanism of the Polygalae Radix-Ziziphi Spinosae Semen (PR-ZSS) drug pair in treating anxiety accompanied by insomnia was explored. Based on the previous analysis results of the chemical components of PR and ZSS by our research group, and combined with databases, the intersection targets of the PR-ZSS drug pair and the disease of anxiety accompanied by insomnia were obtained. GO analysis and KEGG analysis were carried out for the core targets and pathways, and the protein molecular docking method was adopted. Meanwhile, an in vitro cell model of anxiety accompanied by insomnia was established by inducing HT22 mouse hippocampal neuronal cells with 200 μmol/L corticosterone, and the in vitro effect of the PR-ZSS drug pair was verified. The results showed that there were 63 active components in the PR-ZSS drug pair, and 362 targets for treating anxiety accompanied by insomnia. The core targets involved serine/threonine-protein kinase 1 (AKT1), tumor necrosis factor (TNF), etc. Its action may be related to the phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) signaling pathway and others. The molecular docking results showed that the four core components of PR-ZSS had good binding ability with the targets of PI3K, AKT, and brain-derived neurotrophic factor (BDNF). The results of the cell experiment indicated that the medium-dose PR-ZSS group significantly increased the contents of 5-hydroxy tryptamine, norepinephrine, dopamine and γ-aminobutyric acid (P<0.05 or P<0.01 or P<0.001) by increasing the mRNA expressions of PI3K, AKT, BDNF and cAMP-response element binding protein 1 (CREB1), and reducing the production of inflammatory factors interleukin-6 (IL-6), IL-1β and TNF. In conclusion, the mechanism of the PR-ZSS drug pair in treating anxiety accompanied by insomnia may be to regulate the PI3K-AKT signaling pathway and other pathways through components such as coumestrol, oleic acid, sibiricoside A3, and 4-methoxysalicylic acid, and act on targets such as PI3K, AKT and BDNF. This study further proves that the PR-ZSS drug pair can exert anti-inflammatory, sedative, and tranquilizing effects through multiple components, multiple targets, and multiple pathways, and preliminarily clarifies the mechanism of the PR-ZSS drug pair in treating anxiety accompanied by insomnia.