In this work, the effect of trehalose was investigated on the structural stability of the D-Lactate dehydrogenase (DLDH) enzyme. Initially, response surface methodology (RSM) was applied to optimize different process variables for the stabilization of DLDH using trehalose. The stabilization of DLDH with trehalose was examined under various experimental conditions, including trehalose concentration, pH, temperature, and incubation time. The effect of the processing parameters was tested using the RSM method and a central composite design (CCD) model. According to the ANOVA results, there was a close correlation between the predicted and experimental values of the response parameter. The optimum values of trehalose concentration, pH, temperature, and incubation time were found to be 1.25 M, 8, 30 degrees C, and 25 min, respectively, for achieving the maximum activity. Then, the kinetic and thermodynamic parameters of DLDH in the absence and presence of trehalose were examined. The DLDH half-life at 40 degrees C was 28.88 min, while, in the presence of trehalose, the enzyme had a longer half-life (33.01 min) at this temperature. Also, in the end, molecular dynamics (MD) simulations of the DLDH enzyme was performed at the optimum conditions to obtain fundamental insights into the conformational transitions of the DLDH enzyme in the absence and presence of trehalose.