This article investigates the surface chemistry properties of the β-galactosidase monolayer at the air-subphase interface at the vicinity of its substrate, X-gal. We have demonstrated that the β-galactosidase in the monolayer form remained active and performed hydrolysis of the X-gal in the subphase. We investigated the β-galactosidase Langmuir monolayer in absence and presence of X-gal in the subphase of varying concentration of X-gal with the sodium chloride solution. It was found that the limiting molecular area as well as the collapse surface pressure kept on decreasing with the increasing concentration of X-gal. In accordance to the data obtained from the isotherm it was also found that β-galactosidase forms a stable monolayer that does not aggregate at the air-subphase interface. The stability of the monolayer at the air-subphase interface was studied by using compression-decompression cycles with and without X-gal at varying concentration and different surface pressures. The infrared reflection-absorption spectroscopy (IRRAS) and Brewster angle microscopy (BAM) of β-galactosidase Langmuir monolayer was also investigated for pure and mixed β-galactosidase at the air-subphase.