To study the effect of co-administration of Jujuboside A and B (Ju A+B) on sleep, healthy KM mice were given different doses of Ju A+B with a behavioral evaluation of sleep state. Serum levels of key neurotransmitters (5HT, DA, and NE) were measured. The hypothalamus of KM mice was analyzed for differential protein expression using TMT quantitative proteomics, and differential expression protein (DEP) bioinformatics analysis was used to explore potential mechanisms. The result shows that Ju A+B affects sleep by expressing the protein in the hypothalamus. Compared with the control group, the test group showed 10 up-regulated and 139 down-regulated DEPs. The key DEPs were found at the tight junction. Western blot showed reliable alteration of the key DEPs in proteomics. The result of interaction network analysis attributed the potential of Jujuboside to the changes in blood-brain barrier, which provided basic and theoretical data for the efficacy evaluation and mechanism of Jujuboside.