A novel high-throughput method was established for rapid screening of large numbers of Aspergillus niger mutants with high transglucosylation activity by exploiting that yeast can hardly hydrolyze isomaltooligosaccharides (IMO). Supernatants of A. niger fermentation were incubated with Saccharomyces cerevisiae to remove glucose and maltose, and the remaining non-reducing sugars, which is positively correlated with the amount of IMO, the products of transglucosylation reaction, were used as indicator of transglucosidase activity of A. niger and examined by dinitrosalicylic acid assay. Using this method, 15 stains that could convert liquefied cassava starch to IMO more efficiently were selected from 8721 A. niger mutants. Among them, mutant C-6181 strain had transglycosidase activity of 4.61 U/ml (increased by 122% compared to its parental strain) and IMO yield of 83.7%. Taking together, the method is easy, simple, efficient and cheap, and has great application potential in selection of transglucosidase-producing strains used in industrial IMO fermentation.