Transglucosidase (TG), an enzyme produced in Aspergillus sp., could synthesize isomaltoligosaccharide from α-(1→4) linked substrates. In this study, the highest TG producing Aspergillus sp. was selected within six kinds of Aspergillus sp. used as industrial strains in food industry and the optimized TG producing condition was established. Aspergillus niger KCTC6913 showed the highest TG activity on the 15th day. And then, study to investigate the effects of different carbon sources, potato starch, sweet potato starch, corn starch, wheat starch or dextrin, on TG actvity was progressed. As a results, TG activity was showed the highest when treated with potato starch as carbon source. And also, TG activity showed the highest activity cultured with yeast extract added media among three-different nitrogen sources, yeast extract, malt extract or beef extract, respectively. To increase TG activity with aeration on batch fermentation condition, each group were prepared with different media volume as 10%, 20%, 30%, 40% or 60% (v/v) to constant to cultivation straight jar. And the TG activity was increased when the amount of air increased dramatically. C/N ratio was carried out with media made by using various ratio of potato starch and yeast extracts. And then the results showed that TG activity could increased when higher 1 : 1 ratio. Eventually, TG activity was showed higher when using media made with 1.5%(v/v) of potato starch and yeast extract then 1.0%(v/v) of potato starch and yeast extract. Finally, comparative experiment was carried out between optimized media and standard potato dextrose broth in batch fermentation and then their results announced that TG activity could be increased by optimize their cultivation conditions. These results could be suggested the best culture conditions to produce TG from industrial strain such as Aspergillus sp..