The spoilage of cassava by Aspergillus niger strain XJ42 poses significant challenges to food security and industrial biotechnology, necessitating an in-depth understanding of its enzymatic mechanisms. This study aimed to amplify and characterize the α-amylase gene, CDF_Amyl, from A. niger strain XJ42 and evaluate its biotechnological potential. The CDF_Amyl gene was successfully amplified and partially sequenced, revealing a protein consisting of 222 amino acids with a molecular weight of 25.13 kDa and an isoelectric point of 4.17. Sequence alignment demonstrated high homology with α-amylase genes from A. niger (98%) and A. oryzae RIB40 (99%), suggesting evolutionary conservation and potential functional similarities. Kinetic analysis of the enzyme revealed a maximum velocity (Vmax) of 6.90 U/mg protein and a Michaelis constant (Km) of 6.70 mg/ml, indicating its catalytic efficiency. These findings highlight the enzyme’s robust activity and potential applications in biotechnology, particularly in starch hydrolysis for industrial, food, and pharmaceutical processes. The unique properties of CDF_Amyl suggest that it may serve as a valuable biocatalyst for enhancing cassava processing and other starch-based industries. Further studies on its structural and functional properties could facilitate its commercial exploitation in biotechnological innovations.