The prevention of ferrocene leakage from an electrode by physical retention of mediator in a matrix of cellulose acetate membrane is reported. Five types of the cellulose acetate membranes were prepared, containing 1.8%, 5.3%, 8.5%, 20.0% of ferrocene and a membrane containing 1.8% of ferrocene and 0.05 % of Nafion in the matrix. Ferrocene embedded membranes were successfully applied in the construction of a fructose biosensor by immobilization of PQQ-dependent fructose dehydrogenase (FDH). The biosensor comprising a cellulose acetate membrane with 1.8% of ferrocene and 0.05% of Nafion had good stability characteristics, retained almost 40% of the initial response after 8 h of continuous use with an initial sensitivity of 226 nA mM(-1) and response time of 75 s.