The invention discloses a method for extraction separation of a withanolides compound from waste Chinese wolfberry branches. The method comprises the steps of: (1) extracting waste Chinese wolfberry branches with
ethanol, combining the extracted liquid, and recovering
ethanol to obtain a concentrated solution; (2) adding
ethyl acetate into the concentrated solution to perform extraction, recovering the organic phase and then performing concentration to obtain an extract; (3) diluting the extract, loading the diluted extract to a selective macroporous adsorption resin column, conducting
elution with
ethanol, recovering ethanol from the obtained eluent, and conducting
crystallization with ethanol so as to obtain the withanolides compound; (4) loading the withanolides compound
crystal to a
silica gel column, performing
elution with a
mixed solution of n-
hexane and
ethyl acetate, carrying out segmented collection of the eluent, thus obtaining withanolides A and withanolides B respectively in order; and (5) recovering the
solvent from the segmented eluent in step (4), and then performing
crystallization with ethanol so as to obtain a withanolides A
monomer and a withanolides B
monomer respectively. The method provided by the invention has high extraction yield, and the obtained monomers have high purity.