In this work the shift in fluorescence emission spectra of acrylodan, a polar sensitive fluorophore, has been used to characterize the polarity immediately surrounding cytoplasmic (cMDH) and mitochondrial malate dehydrogenase (mMDH) enzyme immobilized within three-dimensional macroporous chitosan scaffolds. The scaffolds were fabricated from solutions of fluorescently tagged enzymes mixed with deacetylated and hydrophobically modified chitosan polymer. Each solution was frozen and then freeze-dried through the process of thermally induced phase separation (TIPS). The blue shift in acrylodan's emission maxima (lambda(max)) revealed a polar shift in the chemical microenvironment surrounding the enzymes when immobilized in a modified as opposed to unmodified chitosan scaffold. These results suggest that the method of hydrophobic modification of native chitosan polymer can be used to control the amphiphilic nature of the chemical microenvironment immediately surrounding the enzyme after it has been immobilized.