The invention discloses a method for separating and purifying acetoin in fermentation liquor. The method includes the step of adding inorganic salt and a weak polar organic solvent are added into the fermentation liquor for extraction, wherein the addition amount of the inorganic salt accounts for 25-125% of the volume of the fermentation liquor, and the volume ratio of the organic solvent and the fermentation liquor is (1-2):1. The weak polar organic solvent is one of ethyl acetate, methyl tertiary-butyl ether, butyl acetate, dimethyl carbonate and diethyl carbonate. By means of the method, an extraction phase containing acetoin hardly contains pigment, residual sugar and organic acid impurities, an extraction solution can be directly rectified, and the scaling phenomenon is not likely to happen to a column reactor in the rectification process. Polarity of the solvent in use is small, the specific heat capacity is low, the upper phase water content is low, energy consumption for recycling is low, and acetoin loss in the recycling process is small. Operation is easy, and the method is an acetoin separation and extraction technology with industrial application prospects.