This study describes the characterization of a novel thermostable α-amylase from a 104.K strain isolated from the Kashkadarya region of Uzbekistan. Phylogenetic analysis revealed that the thermostable α-amylase belongs to glycoside hydrolase family 13 subfamily 5 (GH13_5) and shares high sequence similarity with known α-amylases. Our results demonstrate that the recombinant α-amylase exhibits optimal activity at pH 6.0 and 90 °C, retaining full activity after 30 min at 60 °C. The addition of CaCl significantly enhanced thermostability, with the enzyme retaining more than 95% of its initial activity at 70 °C after 30 min. Our findings indicate that α-amylase from 104.K is a functional, thermostable enzyme with potential industrial applications. This study highlights the commercial significance of thermostable amylases and the need to identify novel, cost-effective, and sustainable sources. The results of this study will contribute to the fields of enzyme applications, stabilizing additives, and genetic engineering of thermostable genes.