Three new polysaccharides (SLP-50, SLP-70, and SLP-90) were fractionated successively by gradient concentrations of ethanol. This study aimed to investigate the initial structural characterization and bioactivities of these sugarcane leaves polysaccharides (SLPs) obtained by ultrasound-assisted extraction. The results, which were further validated by IR spectrometry, revealed that the three SLPs contained uronic acids, proteins, and sulfate groups in addition to carbohydrate. SLP-50 (36.40 kDa), SLP-70 (12.97 kDa), and SLP-90 (3.52 kDa) were acidic heteropolysaccharides mainly comprised of mannose (Man), rhamnose (Rha), glucuronic acid (Glc A), galacturonic acid (Gal A), galactose (Gal), glucose (Glc), xylose (Xyl), and arabinose (Ara), in different molar ratios. SLP-90 exhibited higher antioxidant capacity than SLP-50 and SLP-70. In vitro, all fractions showed significant hypoglycemic potential and antibacterial activity against Gram-positive and Gram-negative bacteria. The IC 50 values of SLP-50, SLP-70, and SLP-90 were 0.11, 0.05, and 0.67 mg/mL on α-glucosidase, respectively, which were significantly lower than that of acarbose (0.80 mg/mL). These findings could provide a reference for developing and applying SLP-based functional foods.