Objective To produce tagatose from fructose with a high conversion rate and to establish a high-yield purification method of tagatose from the reaction mixture. Results Fructose at 1 M (180 g l(-1)) was converted to 0.8 M (144 g l(-1)) tagatose by a three-step enzymatic cascade reaction, involving hexokinase, plus ATP, fructose-1,6-biphosphate aldolase, phytase, over 16 h with a productivity of 9 g l(-1) h(-1). No byproducts were detected. Tagatose was recrystallized from ethanol to a purity of 99.9% and a yield of 96.3%. Overall, tagatose at 99.9% purity was obtained from fructose with a yield of 77%. Conclusion This is the first biotechnological production of tagatose from fructose and the first application of solvent recrystallization for the purification of rare sugars.