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A strain of thermophilic Anoxybacillus flavithermus is characterized bacteriologically and biochemically. Then, the gene responsible for encoding cyclomaltodextrinase (CDase) in this bacterium is isolated, cloned, and overexpressed. The sequence of the CDase is recorded in GenBank with accession number KT633577.1. Biochemical and structural characterization of the enzyme shows that CDase with molecular weight of 72 kDa is active in the optimum temperature and pH of 65 °C and 7.0, respectively and it exists in oligomeric forms. It is observed that α‐cyclodextrin (α‐CD) has higher kcat/km value as compared to starch, glycogen, β‐ and γ‐CD and is therefore a more specific substrate for the enzyme. Also, the addition of metal ions such as K1+ and Na1+ can help to improve the catalytic activity, while Fe2+ and Cu+2 have inhibitory effects on the activity of the enzyme. It is also found that the catalytic activity of the enzyme increases in the presence of 5 mM β‐mercaptoethanol, EDTA, DTT, and PMSF. Thin layer chromatography (TLC) shows that glucose and maltose are the main products of CDase‐catalyzed reaction of α‐, β‐, and γ‐CD hydrolysis. Considering the modeling results, the CDase consists of three domains in which the central domain is the catalytic core containing active site residues. Based on its specificity for various substrates, the new homologue of CDase can be considered as a suitable candidate for industrial applications involving production of maltose and glucose from α‐, β‐, and γ‐CD or a mixture of these compounds in extreme conditions of temperature. New homologue of cyclomaltodextrinase (CDase) in thermophilic Anoxybacillus flavithermus is identified and characterized. It can be considered as a suitable candidate for using in industrial applications involving production of maltose and glucose from α‐, β‐ and γ‐CD or a mixture of these compounds in extreme conditions of temperatures. [ABSTRACT FROM AUTHOR]