Abstract: Background and Objective: Glucoamylase are inverting exo-acting starch hydrolases releasing β-glucose from the non-reducing ends and are used in the manufacture of glucose, fructose syrups and other industrial purposes. The purpose of the study was to optimize the preferable environmental condition for the production of glucoamylase and its characterization from Aspergillus flavus (A. flavus) NSH9. Methodology: The effects of various parameters like carbon and nitrogen sources, temperature, pH, incubation period and other supplements were investigated for the production of glucoamylase. One-way ANOVA and independent samples t-test were used to analyze the data. Results: The maximum production of glucoamylase was observed at 25°C with initial pH 5.0 after 5 days of incubation. Depending on the type and amount of carbon sources, 6% soluble starch was considered as best for the glucoamylase production followed 2% raw sago starch. Yeast extract was considered the best as organic and urea was as inorganic sources of nitrogen. The glucoamylase from Aspergillus flavus NSH9 exhibited optimum activity in a range of pH 4.0-7.0 with a maximum activity at pH 5.0 and the pH stability (more than 95%) ranging from 4-9 at 25°C after 24 h. Conclusion: Glucoamylase from Aspergillus flavus NSH9 have high optimum temperature (70°C) for activity and good thermostablity in the absence of substrate. The crude glucoamylase was also found capable of hydrolyzing the raw starch.