In this study, a co-immobilization process was developed to improve the production of neo-fructooligosaccharides (neo-FOS) by the neo-fructosyltransferase of Penicillium citrinum and to investigate its physicochemical properties. The optimal concentration of CaCl2 for the co-immobilization process was determined to be 0.25 M. In batch production, co-immobilization of whole cells together with neo-fructosyltransferase produced more neo-FOS (108.4 g/L) than did whole cell immobilization (49.4 g/L). In a study on water activity of neo-FOS, we found that the minimal concentration capable of inhibiting bacterial growth was about 42%, and that the minimal inhibitory concentration for fungal growth was about 68%. In terms of pH, neo-FOS was stable in the pH range of 7-10, but was degraded to an increasing extent with decreasing pH, and it was found to be thermally stable at food processing temperatures.