d-Allulose is a low-calorie sweetener and has broad applications in the food, cosmetics, and pharmaceutical industries. Recently, most studies focus on d-allulose production from d-fructose by d-allulose 3-epimerase (DAEase). However, the major blocker of industrial production of d-allulose is the poor thermostability. In this study, site-directed mutagenesis at the interface regions of Dorea sp. DAEase was carried out, and the F154Y/E191D/I193F mutation was obtained. The mutant protein displayed much higher thermostability, with a t1/2 value of 20.47 h (50 °C) and a Tm value of 74.18 °C. Compared with the wild-type DAEase, the t1/2 value at 50 °C increased by 5.4-fold, and the Tm value increased by 17.54 °C. In the d-allulose production from 500 g/L d-fructose, 148.2 g/L d-allulose could be obtained by F154Y/E191D/I193F mutant protein. The results suggest that site-directed mutagenesis at the interface regions is an efficient approach for improving the thermostability of DAEase.