The present invention discloses a process for separation and extraction of 1,3-propylene glycol by fermentation method, which belongs to biochemical separation technical field. A rectification is carried out to separate alcohol in the fermentation liquor, then solids and dissolved protein are separated from the fermentation liquor by composite flocculation of cation type flocculant and non-ion type flocculant. The pH value of the obtained clear liquid is regulated by an acid, and an extraction is performed with an aldehyde. An aldolisation between 1,3-propylene glycol, 2,3-butanediol, glycerine and an aldehyde is carried out to obtain a cyclic acetal to be separated from water phase, salts and other solubility impurity are separated from 1,3-propylene glycol, 2,3-butanediol and glycerine along with the water phase. The acetal hydrolysis applies reacting rectification with solid acid catalyst, can obtain high concentration 1,3-propylene glycol, 2,3-butanediol and glycerine mixture, which is separated again by subsequent common rectification. Excessive aldehyde in the extract phase is recovered by rectification. The invention has the advantages of simple extraction and separation process, low energy consumption, high product recovery, less equipment investment and less land occupation.