This study characterized a novel intracellular α-glucosidase, produced by Debaryomyces hansenii. The enzyme was purified through ultra-filtration, ammonium sulfate precipitation, chromatography Q-Sepharose and Sephacryl S-200 column. SDS-PAGE analysis confirmed the molecular weight of the purified protein to be 125 kDa. The enzyme exhibited maximum activity at pH 4.5and 50 °C and stability at pH 4.0 – 6.0 at 30 – 60 °C. The optimum isomalto-oligosaccharides (IMOs) bio-synthesis temperature using 400 g/L maltose was evaluated to be 35 °C with an optimum enzyme unit determined as 4.34 U g−1. The enzyme exhibits maximum maltose utilization of 78%, with or without inorganic salts, and was inhibited by ZnCl2, CoCl2 and CuSO4. Using maltose, the Km and Vmax values were 7.55 µM and 909 µmol/ml/mg respectively. In 3 L bioreactor, 4.34 U of enzyme produced IMOs with 47% purity utilizing 400 g/L maltose at 35 °C for 14 h. Furthermore, low purity IMOs was passed through cationic ion exchange resins to obtain high purity IMOs by separating glucose and maltose. This pioneering study produced high purity IMOs with 89% purity and 60% yield with greater conversion efficiency using a purified enzyme with a higher concentration of maltose and chromatographic method, which were confirmed by HPLC.