Galacto-oligosaccharides are compounds that are synthesized from lactose containing two to five galactose units and a terminal glucose residue. They are formed from lactose by the transgalactosylation activity of the enzyme β-galactosidase. During the galactooligosaccharides synthesis are formed significant amounts of glucose and galactose, products that are considered reaction inhibitors. This work aims to produce and partly purify galacto-oligosaccharides using sequential fermentation. Galacto-oligosaccharides were produced using Aspergillus oryzae ATCC 11488 by solid-state fermentation (first part); Saccharomyces cerevisiae was used for the selective removal of the monosaccharides from the GOS mixture, (second part). The substrate for the solid-state fermentation was moistened wheat bran with a solution of 2% (w/v) lactose whey permeate. For the production of the galacto-oligosaccharides, a solution of 25% (w/v) lactose whey permeate was used. Enzymatic activity of β-galactosidase was determined using o-nitrophenylgalactopyranoside as substrate. β-galactosidase maximum activity was 0.43 U mL-1 , obtained in 96 hours of solid-state fermentation. Galacto-oligosaccharide yield was 13.7%, area percentage of glucose and galactose was 13.76%, and 7.93% respectively, after 24 hours of fermentation at 40 °C and pH 5.9. After sequential fermentation, 85% of glucose was removed in 48 hours of fermentation and a 63% increase in the area percentage of galacto-oligosaccharides after 60 hours of fermentation. It was shown that sequential fermentation can be used in the partial purification of mixtures of carbohydrates, which facilitates the subsequent separation stages.