The stability of enzymes with no reduction in their catalytic activity still remains a critical issue in industrial applications. Naturally occurring osmolytes are commonly used as protein stabilizer. Apart from increasing the stability and catalytic activity, these osmolytes do not change the structure of enzyme. There are a few general schemes about the stabilization mechanism of these osmolytes but the details of their mechanism have not been found so far. In this study, we investigated the simultaneous effects of sorbitol and trehalose on the activity and structural stability of Pseudomonas cepacia lipase (PCL) using UV–visible, fluorescence and circular dichroism (CD) spectroscopy. In order to trace the refractive index and dielectric constant alterations upon the addition of osmolytes, microenvironment of the enzyme (PCL) was studied by means of SPR technique.The results revealed that osmolytes increased catalytic activity and intrinsic fluorescence intensity of PCL. In the presence of both osmolytes the activity of enzyme is greater than when each of the osmolytes is used individally. Far-UV CD spectra indicated that the secondary structural content of protein has been some what increased upon interacting with these osmolytes.The results of SPR technique indicated none of the above osmolytes could change the dielectric constant of medium considerably. This study revealed the synergy of two osmolytes toward increasing the activity and stability of enzyme.