Ionic liquids (ILs) have emerged as an alternative to conventional organic media due to their high thermal and chemical stability, negligible vapour pressure, non-flammability and easy recycling. In this context, this work assesses the catalytic activity of a β-galactosidase from Bacillus circulans ATCC 31382 (β-Gal-3-NTag) in the synthesis of β-(1→3)-galactosides using different ILs. A noticeably increase in activity, retaining total regioselectivity was found in the synthetic behaviour of B. circulans β-galactosidase in ILs as co-solvents and using a 1:5 molar ratio of donor (pNP-β-Gal):acceptor (GlcNAc or GalNac). Yields up to 97% of β-(1→3) with different ILs were found. These reactions take place without noticeable hydrolytic activity and with total regioselectivity, representing a considerable improvement over the use of aqueous buffer or conventional organic solvents. Furthermore, reaction scaling up and IL recovery and recycling are feasible without losing catalytic action. Molecular modelling studies performed predict a three-dimensional interaction at the active centre between the acceptor and the water–IL mixture, which could explain the results obtained.