Dextranase has extensive applications in the food, pharmaceutical, and chemical industries. However, enzyme production by the wild-type strain is generally low and few studies have conducted structural analysis of dextranase. This study describes an efficient and stable mutagenesis method for producing dextranase and the relationship between the enzyme structure and enzymatic properties before and after mutagenesis. Wild-type Chaetomium globosum was mutated by combined atmospheric and room temperature plasma treatment and ethyl methyl sulfone application. Mutant strains were screened for optimal dextranase production ability and genetic stability. The maximum yield of dextranase reached 824.73 U/mL. Dextranase produced by the mutant strain displayed the same optimum pH and temperature values of 5.5 and 60 degrees C, respectively, as dextranase produced by the wild-type strain. However, dextranase from the mutant strain showed greater heat stability at 70 degrees C. Furthermore, the three-dimensional structure of the enzyme changed slightly after mutagenesis, with the amino acid residue at position 578 changing from His to Leu. This change in the three-dimensional structure affected the properties of dextranase. Finally, dextranase significantly inhibited the growth of Streptococcus mutans. The results indicated dextranase from Chaetomium globosum would give potential implications in the medical, prevent dental caries and food industries.