The present study was conducted to optimized culture conditions and environmental factors that enhance cyclodextrin glucanotransferase (CGTase) production by a previously isolated, new alkaliphilic Amphibacillus sp. NPST-10. Production of CGTase was growth-associated, with enzyme synthesis starting during the early exponential-growth phase and increasing as the cells grew exponentially, reaching the highest concentration levels (0.6 U/ml) at the end of the stationary growth phase (38 h) and remaining constant during prolonged incubation up to 60 h. While mono- and disaccharides supported Amphibacillus sp. NPST-10 growth, maximal CGTase production was detected using various types of starch as the main carbon source, with maximum enzyme production observed with 1.75% soluble starch. Organic nitrogen sources enhanced CGTase production, with a mixture of peptone, yeast extract, and casamino acids (1.2% w/v) supporting maximal production. In addition, Amphibacillus sp. NPST-10 was able to grow in a wide range of NaCl concentrations up to 15%, with maximal growth and CGTase production at 6% and 3%, respectively, indicating the halophilic nature of this bacterium. Furthermore, maximal Amphibacillus sp. NPST-10 growth and CGTase production were detected at pH 9.5 and 9.0, respectively, indicating that Amphibacillus sp. NPST-10 is alkaliphilic in nature. Moreover, maximum enzyme production was observed at 35°C with aeration by shaking at 150 rpm. Addition of Zn²⁺, Cu²⁺, Co²⁺, or Ba²⁺ ions led to reductions both in bacterial growth and CGTase production, particularly at a high metal concentration (10 mM), by approximately 83.6%, 72.7%, 57.6%, and 31.6%, respectively. However, inclusion of Ca²⁺ ions in the culture medium led to a significant increase in CGTase production of approximately 16%. These results indicated that optimizing the culture medium and environmental conditions increased Amphibacillus sp. NPST-10 CGTase production by approximately two fold.