The mixture of both Dahlia (Dahlia pinnata) tubers and Chicory (Cichorium intubus) roots has the potential to be an effective and cost-efficient substrate for Exo-inulinase production more than each alone. Five Aspergillus species were also examined for maximum production of Exo-inulinase activities when grown on aforementioned mixture. Aspergillus terreus was the potent on a ratio of (5:1 w/w Dalia: Chicory, respectively) producing the maximum levels of inulinase at 45 ºC, pH 7.0, seven days of incubation, 50 % moisture and peptone as the best nitrogen source. With 70.23 purification fold, inulinase was purified to homogeneity. Studying of inulinase thermal stability for 2 hours at 60 °C revealed a significant reduction in the free enzyme activity when compared to the immobilized one. With a 75.65 % immobilization yield, inulinase was successfully immobilized. The sodium alginate beads passed the reusability test after 10 cycles and 20 days of storage at 4°C. These findings imply that inulinase in its immobilized form could be a potential candidate for pure fructose syrups production. It was also found that ethanol production by both free and immobilized inulinase was achieved using two processes; direct conversion and Sequential hydrolysis and fermentation of inulin into ethanol using Saccharomyces cerevisiae for fermentation sugars into ethanol. To the best of our knowledge, there is the first report about immobilized inulinase production by Aspergillus terreus using Dalia tubers and Chicory roots mixture and its usage in ethanol production.