Longan pulp polysaccharides were extracted with hot water (LP-H), superfine grinding (LP-S) and superfine grinding-assisted enzymatic treatments (LP-SE). The yields, physicochemical properties and prebiotic activities of polysaccharides were investigated. Compared with LP-H and LP-S, the yield, sugar content, solubility, arabinose and mannose percentage of LP-SE increased while its apparent viscosity, particle size, Mw and glucose percentage declined. Three LPs contained similar glycosidic linkage of →3)-α-l-Araf-(1→, →3,6)-β-d-Galp-(1→ and α-l-Rhap(l→, while they each contained specific glycosidic linkage of →4)-β-d-Glcp(l→, →4)-β-d-Galp-(1→ and →5)-α-l-Araf-(1→ in LP-H, LP-S and LP-SE, respectively. Moreover, LP-SE exhibited stronger stimulation than LP-H and LP-S on the proliferation of Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus fermentum and Leuconostoc mesenteroides. The results indicated three extraction methods had some effect on chemical composition and structure of polysaccharide. LP-SE extracted by superfine grinding-assisted enzymatic treatment exhibited the highest prebiotic activities, which have a great potential in applying in functional food and medical industry.