The invention relates to immobilized cells capable of producing fructooligosaccharides and a preparation method and application of the immobilized cells. The preparation method comprises the following steps: (1) adding glutaraldehyde into bacterium suspension of fructooligosaccharide producing bacteria, reacting and washing so as to obtain crosslinking bacteria; (2) preparing crosslinking bacterium suspension from the crosslinking bacteria, mixing with immobilizing liquid, adding the mixed feed liquid into a calcium chloride solution, hardening, and performing solid-liquid separation, thereby obtaining the immobilized cells. According to the method disclosed by the invention, the cells are first treated by glutaraldehyde and subjected to PEG600 (polyethylene glycol) pore forming and calcium alginate gel embedding, calcium chloride is supplemented in the saccharification process, and finally the immobilized cells which are high in enzyme activity stability and high in mass transfer efficiency and have the enzyme activity of higher than 70% when continuously used for 3 months are obtained.