Objective： This study was aimed to improve the thermal stability of agarase from Microbulbifer sp. AG1 by rational design. Methods： According to the analysis of PoPMuSiC online analytical tool， the mutation site where the stability of agarase could be improved was selected. The mutant gene was obtained by means of overlapping extension PCR. After induction， expression and purification， the mutant enzyme was characterized. Results： The mutant D136N with improved thermostability was obtained after screening. After incubation at 60 ℃ for 1 h， the mutant could maintain about 87% of residual activity， while the wild-type agarase could retain 19% of residual activity. The mutant enzyme D136N had a better thermal stability. Three-dimensional modeling showed that Asp136 formed two hydrogen bonds with Asn255 and one hydrogen bond with Tyr282 in the wild-type agarase. In the mutated enzyme， Asn136 formed three hydrogen bonds with Asn255， one hydrogen bond with Tyr282， and one hydrogen bond with Ala134. Therefore， the amino acid site of 136 was able to form more hydrogen bonds with its surrounding amino acid residues in the mutant of D136N. Conclusion： The mutant D136N with improved thermostability was obtained by the rational design. It is important to improve the enzymatic properties， to expand the application of agarase， and to study the relationship between the structure and function of agarase.