This invention relates to a method for separating and purifying 1,3-dihydroxypropane from microbial fermentation liquor, comprising: after the sterilization and dehydration of fermentation liquor, add in 1-pentanol or isoamyl alcohol, n-butanol and n-hexanol, then remove the sediment in the fermentation liquor by filtration or settlement followed by washing; add in appropriate amount of glycerol or 1,4-butylene glycol to the supernatant to recover 1-pentanol or isoamyl alcohol, n-butanol and n-hexanol, by decompression or normal pressure distillation, so as to produce high-purity 1,3-dihydroxypropane, with more than 95% recovering rate. This invention is characterized of simple separation process, mature technique, low separation cost, and little additional introduced agent with easy recovery. Besides, the crystallization sediment in the fermentation liquor can be efficiently separated, which overcomes the problems of foaming, agglomerating and pipe clogging during distillation, so as to realize continuous operation.