Provided is a separation and purification method for vancomycin hydrochloride of high purity. The method comprises the following steps: (1) obtaining a vancomycin hydrochloride solution from a crude vancomycin product by ion exchange chromatography and obtaining a concentrate by nanofiltration desalination and concentration； (2) adjusting the concentrate with a hydrochloric acid solution and then performing a column chromatography using a reverse chromatography column for the adjusted concentrate； (3) collecting the chromatographic solution of vancomycin to obtain a mixed chromatographic solution； (4) adjusting the mixed chromatographic solution, and separating the solution and the salts by nanofiltration desalination and concentration to obtain a concentrate； and (5) obtaining a vancomycin dry powder with a chromatographic purity of up to 99% and a pure white appearance by dehydrating and drying the concentrate of step (4), or by solvent crystallization or salting-out crystallization.
