Prolonged sleep deprivation impairs brain function and increases the risk of mental health disorders. Cichorium intybus L. Oligo-polysaccharides (JSO), bioactive compounds derived from chicory, belong to the category of food-medicine homologous substances, possess gut microbiota-modulating and anti-inflammatory properties, and serve as a natural prebiotic, having significant research value in food science. This research examined the anxiolytic properties of JSO in a murine model subjected to chronic sleep deprivation (CSD) stress and explored the mechanisms behind this effect, providing experimental evidence for the development of Cichorium intybus L. as a functional food. Specific pathogen-free (SPF) KM male mice were allocated at random into six experimental groups: the control group, the CSD model group, the diazepam (10 mg/kg) group, and the JSO treatment groups at low (50 mg/kg), medium (100 mg/kg), and high (200 mg/kg) doses. Following 3 weeks of CSD, anxiety-like behaviors were assessed using the open field test, elevated plus maze test, light-dark box test, forced swim test, and marble-burying test. To analyze the composition of gut microbiota, 16S rRNA sequencing was employed, while protein expression in the BDNF, PI3K/AKT/mTOR, and NLRP3 inflammasome pathways was detected by Western blot. Behavioral analysis indicated that JSO (at doses of 100 and 200 mg/kg) markedly enhanced both the time allocated to open arms and the number of entries into open arms in the elevated plus maze test (p < 0.05). JSO (at doses of 50 and 200 mg/kg) significantly elevated transitions in the light-dark box test (p < 0.05), all JSO doses drastically cut down marble-burying behavior (p < 0.001, p < 0.01, p < 0.01). The 16S rRNA sequencing indicated that JSO intervention increased Bacteroidetes abundance while reducing Actinobacteria. Western blot analysis demonstrated that JSO significantly downregulated the ratios of p-mTOR/mTOR, p-PI3K/PI3K, p-AKT/AKT, BAX/BCL-2, as well as the expression levels of NLRP3, ASC, Caspase-1, and IL-6 proteins (p < 0.05), while upregulating hippocampal BDNF (p < 0.05). These results indicate that JSO ameliorates CSD-induced anxiety-like behaviors by restoring gut microbiota homeostasis, regulating the BDNF-PI3K/AKT/mTOR and BAX/BCL-2 apoptosis pathways, and suppressing NLRP3 inflammasome-mediated neuroinflammation.