The separation of 1,3-propanediol (1,3-PD), butyric acid (BA) and acetic acid (HAc) from the fermentation broth was studied by two-step salting-out extraction. In the first salting-out extraction, the partition coefficient and recovery of BA reached 42.21 and 96.42%, respectively, under the optimal condition of 25 wt% NaH2PO4/30 wt % n-butyl acetate. Subsequently, 91.28% of BA in the organic phase could be recovered through back-extraction when sodium carbonate solution was mixed with organic phase with initial phase ratio of n-butyl acetate solution to alkaline solution being 2:1, in which the molar ratio of sodium carbonate to organic acids was 3:5. Finally, 50% (v/v) ethanol (95%) was added to the bottom phase for the second step salting-out extraction. And the partition coefficient and recovery of 1,3-PD were 9.40 and 95.50%, and those of HAc were 7.46 and 94.40%, respectively. All the cells and most of the proteins (97.16%) could be removed. Effective separation of 1,3-PD from BA was realized by two-step salting-out extraction, which provides a potential method for separation of 1,3-PD, BA and HAc on an industrial scale.