The invention discloses a preparation method and application of high-molecular-weight deacetylated hyaluronic acid. The preparation method comprises the following steps: dissolving hyaluronic acid in anhydrous hydrazine containing 1% of hydrazine sulfate, reacting in a deoxidized nitrogen protective atmosphere at 60 DEG C for 72 hours, precipitating the reaction product with cold ethanol, dissolving the precipitate in 5% glacial acetic acid, adding 0.5M of iodic acid, standing at 4 DEG C for 2 hours, and adding excessive iodic acid into 57% hydriodic acid; and carrying out ether extraction to remove generated iodine, neutralizing, and carrying out freeze-drying to obtain the high-molecular-weight deacetylated hyaluronic acid. The zwitterion hyaluronic acid polysaccharide disclosed in the invention is used for immunological diseases, and has the advantages of obvious curative effect and no side effect.