In this paper, a combination of hot-pressure, enzymatic hydrolysis and membrane separation process is used for efficiently and environmentally friendly extraction of chondroitin sulfate (CS) from large hybrid sturgeon cartilage, namely, HPCS. The recovery and yield of CS were 93.68% and 36.47% under the optimized conditions. Fourier transform infrared (FT-IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography (HPLC) indicated that the HPCS was composed of monosulfated disaccharides in position 6 and 4 of the N-acetyl-D-galactosamine (58.38% and 27.34%, respectively) and nonsulfated disaccharide (14.29%), which was similar to the composition of CS extracted by dilute alkali-enzymatic hydrolysis-chemical precipitation from large hybrid sturgeon cartilage (SCS). The wound healing results indicated that HPCS could promote cell migration and proliferation, alleviate inflammation and facilitate angiogenesis, which results in its excellent wound treatment activity. These results provide theoretical and practical significance for the production and application of chondroitin sulfate.