This work describes neo-fructooligosaccharides (neo-FOSs) production using the immobilized mycelia of Penicillium citrinum. Some critical factors were evaluated to optimize maximal production of neo-FOS. Optimal alginate and cell concentrations were determined to be $1.96\%$ alginate and $7.17\%$ cell, respectively, by statistical analysis. The optimal concentration of $CaCl_{2}$, which is related to bead stability, was determined to be 2 M. It was possible to increase the neo-FOS production by adding 15 units of glucose oxidase to the batch reaction. By co-immobilizing cells and glucose oxidase, neoFOS productivity increased $123\%$ compared with the whole-cell immobilization process. Based on the results above, a co-immobilization technique was developed and it can be utilized for large-scale production.