This article investigates the production and optimization of levansucrase using synthetic medium by Zymomonas mobilis NRRL B-14023. Response surface methodology was used to investigate the effects of three different fermentation parameters on levansucrase enzyme production. Maximum levansucrase activity of 13.3 mu mol glucose/min was obtained at the optimum levels of process variables (pH 4.90, initial sucrose concentration 159.01 g/L and fermentation temperature 30.3 degrees C). Response surface methodology was found to be useful in optimizing and determining the interactions among process variables. The purity of levansucrase used in enzyme assays were analyzed by SDS-PAGE and a dominant protein band of size around 47 kDa was obtained. The sequence of the produced enzyme was determined by peptide mass fingerprinting. The presence of kestose in the reaction mixture showed that Z. mobilis levansucrase can produce fructooligosaccharides. This study fulfills the lack of mathematical and statistical approaches in optimizing the levansucrase production by Z. mobilis.