The purpose of this study is to show how disaccharides differ in their ability to protect lyophilized β-galactosidase from enzymatic activity loss and secondary structure changes during storage. β-galactosidase was lyophilized with trehalose, sucrose, cellobiose or melibiose at 2:1, 20:1 and 40:1 excipient/protein weight ratios, and stored up to 90 days at 45 °C. Protein enzymatic activity was studied using o-nitrophenyl-β-D-galactopyranoside cleavage test, and its secondary structure in lyophilizates analyzed using Fourier transform infrared spectroscopy. The crystallization tendencies, glass transition temperatures and water contents of lyophilizates were evaluated using x-ray powder diffractometry, differential scanning calorimetry and thermogravimetry, respectively. The enzymatic activity of β-galactosidase decreased more slowly in lyophilizates containing trehalose or melibiose at 2:1 excipient/protein weight ratio when compared to those containing sucrose or cellobiose. Similar behavior was observed when analyzing the protein's secondary structure in lyophilizates. In 20:1 and 40:1 excipient/protein weight ratio lyophilizates the decrease of enzymatic activity was less dependent on the excipient, but activity was always amongst the highest in melibiose lyophilizates. Melibiose was shown to be effective in protecting lyophilized β-galactosidase during storage. The protein secondary structure was shown to change at comparable rate in lyophilizates as its enzymatic activity after rehydration.