The β-galactosidase is important in food and pharmaceutical industry, as it hydrolyses lactose into glucose and galactose. It is used on large scale to improve dairy product characteristics and as food supplement by lactose intolerants. The obtention of this recombinant enzyme, through Escherichia coli expression, allows higher amounts of β-galactosidase to be produced involving different purification steps. The objective of the present work was to determine the chromatographic conditions of recombinant β-galactosidase enzyme obtention. E. coli strain BL21 (DE3), transformed with the β-galactosidase gene from Kluyveromyces sp., was cultured in 2 liter bioreactors under predetermined conditions, the cells were disrupted using a French press. After DNA precipitation with streptomycin sulphate, the protein content was submitted to high performance liquid chromatography. Ion exchange techniques (HiPrep Q HP chromatographic column) and hydrophobic interaction (Source 15 PHE column), were tested using the ÄKTA system. The best separation and recovery conditions of the enzyme, were obtained using the HiPrep Q HP column. The complete purification of the β-galactosidase wasn’t achieved, but in all tested conditions the protein showed enzymatic activity.