Surface pressure and surface potential-area isotherms were used to characterize the lysozyme Langmuir monolayer. The compression - decompression cycles and stability measurements showed a homogeneous and stable monolayer at air-water interface. Salt concentration in the subphase and pH of the subphase were parameters controlling homogeneity and stability of the Langmuir monolayer.In situ UV-vis and fluorescence spectroscopies were used to verify the homogeneity of the lysozyme monolayer, and to identify the chromophore residues in the lysozyme. Optimal experimental conditions were determined to prepare a homogeneous and stable lysozyme Langmuir monolayer.