The invention discloses a method for simultaneously preparing 1,3-propylene glycol and 2,3-butylene glycol from glycerol by using microbe employing prior-stage anaerobic and later-stage aerobic two-stage fermentation process, which belongs to the field of the bio-chemical engineering technology. In the fermentation process, the seed liquid is added in the primary fermentation medium containing industrial glycerol or glycerol fermentation liquid, after 3-5 hours, the mixed liquid of glycerol and glucose is added in the form of the flow. The product concentrations of 1,3-propylene glycol and 2,3-butylene glycol are controlled by adjusting the air flow in the two-stage fermentation process and controlling the fermentation temperature. When the fermentation process is over, 1,3-propylene glycol and 2,3-butylene glycol are separated and purified by desalting, distilling, and vacuum rectification. The preparing method can improve the fermentation concentration and the yield of 1,3-propylene glycol and the production intensity, at the same time, 2,3-butylene glycol with higher concentration can be obtained with high raw material availability and low cost.