This study aimed to investigate the effects of ultrasound-assisted extraction (UAE) on the physicochemical properties, biological activities, and intestinal flora regulatory capacity of jackfruit polysaccharides (JPs). Under optimized UAE conditions (liquid-to-solid ratio 30 mL/g, extraction time 30 min, power 90 W), the yield of JP reached 8.70 ± 0.11%. Compared with hot-water-extracted jackfruit polysaccharides (HAE-JPs), ultrasonic-assisted extracted jackfruit polysaccharides (UAE-JPs) exhibited a lower molecular weight, a smaller particle size, and a significant 11.5-fold increase in galacturonic acid content. Structural analyses confirmed that UAE-JPs retained a triple-helix and highly branched conformation but with enhanced exposure of acidic monosaccharides. These structural modifications contributed to superior antioxidant activity and enzyme inhibition ability, demonstrated by its lower IC₅₀ values against DPPH, ABTS radicals, and α-glucosidase. Crucially, in vitro fecal fermentation revealed that UAE-JPs and HAE-JPs differentially modulated the gut microbiota. UAE-JPs preferentially promoted the proliferation of Lactobacillus (an increase of 27.04%) and Bifidobacterium, facilitating short-term acidification. In contrast, HAE-JPs enriched butyrate-producing bacteria like Clostridium (increase of 18.56%). Both polysaccharides significantly inhibited the growth of Fusobacterium (a decrease of 5.23%) related to cancer. Consequently, this study establishes UAE as a green and efficient technique capable of not only modifying the structure of JPs but also precisely tailoring their prebiotic functionality, which ultimately demonstrates the potential of UAE-JPs as a functional food ingredient with enhanced bioactivity.