beta-Glucuronidase has been widely used in improving the efficacy of the natural glucuronides, but the poor thermostability largely impedes its industrial application. In this study, the thermostability of beta-glucuronidase from Penicillium purpurogenum Li-3 (PGUS-E) was enhanced by rationally mutating key residues within the catalytic domain based on in-depth structure analysis and sequence alignment. Three mutants F292L/T293K, S35P, R304L were obtained that showed significantly improved thermostability. PGUS-E showed a two-phase thermal deactivation process, and the thermal deactivation constants k(1) and k(2) were solved separately in each phase. The mutation of F292L/T293K and S3SP contributed more to the maintenance of the enzyme stability in the first deactivation phase, with k(1) decreased by 1 magnitude compared to that of wild-type. Meanwhile, the mutation R304L mainly took effect in the second deactivation phase with the lowest k(2) of 0.0021 min(-1). In addition, mutant F292L/T293K showed 6.4 times higher k(cat)/K-m than wild-type. The MD simulation indicated that the improved thermostability of the three mutants was due to a unique C-terminal fixing effect (F292L/T293K), proline effect (S35P) and hydrophobic interaction (R304L). This study not only promotes the industrial application of beta-glucuronidase but also provides new insight into the interplay between structure and stability of beta-glucuronidase.