Lotus leaves are often discarded as byproducts in the lotus industry. Polysaccharides are regarded as one of the essentially bioactive components in lotus leaves. Therefore, in order to promote the application of lotus leaves in the functional food industry, the deep eutectic solvent (DES) assisted extraction of polysaccharides from lotus leaves (LLPs) was optimized, and structural and biological properties of LLPs extracted by DES and hot water were further investigated. At the optimal extraction conditions (water content of 61.0% in DES, extraction temperature of 92 °C, liquid-solid ratio of 31.0 mL/g and extraction time of 126 min), the maximum extraction yield (5.38%) was obtained. Furthermore, LLP-D extracted by DES and LLP-W extracted by hot water possessed the same sugar residues, such as 1,4-α-D-GalAp, 1,4-α-D-GalAMep, 1,3,6-β-D-Galp, 1,4-β-D-Galp, 1,5-α-L-Araf, and 1,2-α-L-Rhap, suggesting the presence of homogalacturonan (HG), rhamnogalacturonan I and arabinogalactan in both LLP-W and LLP-D. Notably, LLP-D was much richer in HG fraction than that of LLP-W, suggesting that the DES could assist to specifically extract HG from lotus leaves. Additionally, the lower molecular weight and higher content of uronic acids were observed in LLP-D, which might contribute to its much stronger in vitro antioxidant, hypoglycemic, and immunomodulatory effects. These findings suggest that the optimized DES assisted extraction method can be a potential approach for specific extraction of acidic polysaccharides with good bioactivities from lotus leaves for applications in the functional food industry.