In the present study, ultrasonic-assisted extraction (UAE) of polysaccharides from Orchis chusua D. Don (salep) was optimized by response surface methodology (RSM) combined with a Box-Behnken design (BBD), the optimum conditions were determined to be: ultrasonic power of 390 W, extraction temperature of 60 °C, extraction time of 50 min and water/raw material ratio of 40 mL/g. Based on it, 48.30 ± 0.6% yield of crude polysaccharide was obtained. After purification, the chemical compositions, physicochemical and structural property was investigated based on the GC-MS, TGA, XRD, SEM and Congo red test. The results showed that USP-U and USP-N were hetero-polysaccharides, mainly composed of mannose and glucose, and the average molecular weight was 25.57 kDa and 11.64 kDa, respectively. FT-IR spectra and NMR results indicated that the presence of α-linkage glycopyranose via typical peaks. Furthermore, reducing power and antioxidant activities of both purified fractions showed a relatively high ability to scavenge free radicals. Lastly, the cytotoxicity assay showed that purified polysaccharides have no obvious cytotoxicity against RAW267.4 and L6 cells at their effective concentration and had potent anti-inflammatory activity in a concentration-dependent manner. Salep polysaccharide is expected to be developed as a new efficacy factor in the pharmaceutical and food industry.