© 2022. International Food Research Journal. All Rights Reserved.β-galactosidase is an enzyme that catalyzes the hydrolysis of lactose, a disaccharide, into glucose and galactose. β-galactosidase is a commercially essential enzyme for its catalytic properties and is widely used in various biotechnology processes. In this study, optimum production conditions were studied to produce the highest level of β-galactosidase using Kluyveromyces marxianus isolated from kefir grains, and changes in some fermentation metabolites during enzyme production were determined under optimum conditions. The pH, temperature, and incubation time were optimised to produce of β-galactosidase. The pH (4.0, 7.0, 8.0), temperature (25 - 37°C), and incubation time (0 - 60 h) were evaluated in the ranges. The highest specific enzyme activity was 47.31 U/mL at the end of 48 hour-incubation at 200 rpm, at pH 8.0, and 30°C with 2% (v/v) inoculation rate. A high-performance liquid chromatography system determined the changes in lactose, glucose, galactose, lactic acid, and ethanol concentrations at the optimum fermentation conditions by taking samples from liquid fermentation medium during enzyme production under these conditions. The amount of glucose and galactose formed by the lactose fermentation metabolism of K. marxianus decreased during the fermentation. The ethanol concentration reached its highest concentration (18.44 g/L) at the 36th hour of fermentation. When fermentation metabolites were examined, β-galactosidase and K. marxianus metabolism treatment indicated remarkable findings.