The invention provides a chromatographic separation device, and a method for separating a vitamin C mother liquor by using the same. The chromatographic separation device comprises a feed tube, discharge tubes and a plurality of separators connected in series, a distributor, a separation material layer and a collector are sequentially arranged in the tank body of every separator from top to bottom, the distributors are connected with the feed tube, and the collectors are communicated with the discharge tubes; the outlet of the collector of every separator is communicated with the inlet of thedistributor of the next separator; and the outlet of the collector of the last separator is communicated with the inlet of the distributor of the first separator. The vitamin C mother liquor is subjected to decolorizing, filtration, concentration, ion exchange and secondary filtration, then is conveyed into the chromatographic separation device, and is separated to obtain gulonic acid and vitaminC. The chromatographic separation device, and a method for separating a vitamin C mother liquor by using the same have the advantages of short process route, low running cost, and great reduction of the sewage discharge amount.