This paper describes optimization method that combines the Plackett-Burman design, a factorial design and the response surface method, which were used to optimize the medium for the production of L-asparaginase by Serratia marcescens SB08. Four medium factors, from out of 11 medium factors, were screened by Plackett-Burman design experiments and subsequent optimization process to find out the optimum values of the selected parameters using central composite design was performed. Sucrose, peptone, KH2PO4 and incubation time were found to be the best medium factors for the optimization of L-asparaginase production and central composite design experiments indicated the optimal concentrations of sucrose 12.50 g/l, peptone 4.5 g/l, KH2PO4 4.0 g/l and incubation time 51h. The combined optimization method described here is the effective for screening medium factors as well as determining their optimum levels for the production of L-asparaginase by Serratia marcescens SB08.