Four commercial 1812 spiral wound nanofiltration membranes were firstly applied to examine the separation performance of sugar solutions in total recycle mode of operation. Membrane NF-3 (supplied by Sepro Co. (USA)) with molecular weight cut-off of 800–1000 Da was selected for separation of a commercial galactose-oligosaccharide (GOS) mixture with low content of oligosaccharides in constant volume diafiltration (CVD) process at 50 °C and 6 bar pressure. The choosing of membrane and operation conditions for CVD process was based on a compromise between the permeate fluxes and apparent rejections of the sugar solution. In CVD course, the concentration of various sugars and the relationship between the yield and purity of oligosaccharides predicted well with mathematical models for the following situation: when the volume of every removing batch of permeate is equal to the volume of tank, the increased rate of sugar rejections is less than 8% and the decreased rate of tank concentrations is less than 15%. 90.5% monosaccharide and 52.5% lactose in the mixture were removed with NF-3 membrane in this study, and the oligosaccharides purity of 54.5% was achieved (1.5 times of the raw material), obtaining oligosaccharides yield of 70.0%.