During the fructose and polymerization to synthesize levan, smaller fructooligosaccharide (FOS) molecules are produced. FOS can also be obtained by levan hydrolysis. Three-phase partitioning (TPP) is a separation technique that has been used for polysaccharide precipitation and gathers t-butanol and ammonium sulphate to exclude the polymer from the aqueous solution. In this work TPP was tested to separate levan and FOS from aqueous solution. The FOS used was obtained from Zymomonas mobilis levan acid hydrolysis and fractionation with ethanol. The yield of low TPP fractions was higher than those obtained from the native levan. The F-90 exhibited a higher yield than other fractions. However, when applying the TPP technique to lightest fraction not precipitated by ethanol at 90% (F>90), the intermediate phase was not possible to be visualize. These results have potential application because they show that by using the levan TPP separation it is possible to separate low-molecular weight sugar. (C) 2010 Published by Elsevier Ltd.