This invention belongs to the field of biochemical engineering and relates to a method of cyclic utilization of water during separation of succinic acid made by fermentation. This invention uses water from separation process for aerobic growth of E. coli AFP111 and production of succinic acid by anaerobic fermentation, obtaining final succinic acid concentration of 55 g/L and yield of 91.6%. Compared with results of fermentation using culture medium prepared from tap water, succinic acid concentration and productivity increased by 8.5% and 8.46%, respectively. An outstanding advantage of this invention is recovery and utilization of evaporated water during separation of succinic acid, realizing cyclic use of water during industrial production of succinic acid, which is an environment-friendly process. Also, as evaporated water generated during separation of succinic acid contains small amount of organic acids such as acetic acid and formic acid, if this water is used for aerobic growth of thalli, the small amount of organic acids contained therein can be used as gluconeogenesis carbon source, improving activity of some key enzymes in cell and favoring succinic acid production by anaerobic fermentation of thalli.