The high temperature induces conformational changes in β-glucosidase, making it inactive and limiting its application field. In this paper, the effect of trehalose on the thermostability of β-glucosidase from low-moisture Hevea brasiliensis seeds was investigated. The results showed that the residual enzyme activities of β-glucosidase supplemented with trehalose after high-temperature treatment were significantly higher than that of the control group. The improvement of thermostability could be explained by low-field nuclear magnetic resonance (LF-NMR) and molecular dynamics (MD) simulations at the molecular level. Moreover, adding trehalose increased the water activity and water content of β-glucosidase, leading to a more stable conformation. Trehalose replaced some water and formed a stable network of hydrogen bonds with protein and surrounding water. The glass formed by trehalose also reduced molecular movement, thus providing good protection for enzymes.