Equilibrium unfolding of a 69-kDa monomeric Escherichia coli maltodextrin glucosidase (MalZ) was studied using intrinsic and extrinsic fluorescence spectroscopy. The unfolding transition of MalZ followed a three-state process, involving the formation of a stable intermediate state having more exposed hydrophobic surface. It was found that the protein structure can be easily perturbed by low concentration of guanidium hydrochloride (GdnHCl) and, at a GdnHCl concentration of 2 M, MalZ was denatured completely. The active site of the protein also has been proved to be sensitive to a low concentration of GdnHCl since MalZ deactivated at 0.5 M GdnHCl completely. The surface hydrophobicity and ANS-binding site of the protein have been determined to be 150.7 and 0.24, respectively. Perhaps the formation of the stable unfolding intermediate, having higher surface hydrophobicity, may be one of the reasons for aggregation of MalZ and its recognition by chaperonin GroEL during the assisted folding pathway.