The invention relates to a method for preparing gentiooligosaccharide by using beta-1,6-glucanase and an application of the method and belongs to the technical field of genetic engineering and fermentation engineering. The invention firstly provides beta-1,6-glucanase TcBgn with transglycosylation activity on gentiobiose, the beta-1,6-glucanase TcBgn can transform glucose into gentiooligosaccharide under the condition of relatively small additive amount of enzyme, and the transformation rate is high, so that the production cost can be significantly reduced. The gentiooligosaccharide is prepared by double-enzyme compounding of the beta-1,6-glucanase and beta-glucosidase, and the glucose is transformed into the gentiobiose and gentianose by using a double-enzyme compounded system, so that the transformation rate is high, and the proportion of the gentianose in a product is significantly improved. Therefore, the method for preparing the gentiooligosaccharide by a single enzyme of the beta-1,6-glucanase or double-enzyme compounding has good industrial application value.