Glutathione peroxidase (GPX) is a selenoenzyme that protects the biomembrane and other cellular components against oxidative damage. The selenium-containing single chain Fv fragment of monoclonal antibody 2F3 (SescFv2F3 ) is a kind of GPX mimic and it has a wide clinical applications because of its high activity and low antigenicity. Se-scFv2F3 is generated by the chemical modification of the single chain Fy fragment of monoclonal antibody 2F3 ( scFv2F3 ), which can be expressed in E. coli. In this article, the effect of chemical chaperones, such as glycerol, glucose, and β-cyclodextrin added to the culture medium, on the expression of soluble scFv2F3 was investigated. The expression level was evaluated by the determination of soluble scFv2F3 contents in the whole cell lysates.The results suggest that both glycerol and β-cyclodextrin greatly increase the expression level of soluble scFv2F3, and β-cyclodextrin is found to be more effective compared with glycerol. Glucose has a slight effect on the expression level of soluble scFv2F3. This is the first example, wherein β-cyclodextrin has been used as a chemical chaperone during the cell culture to improve the expression level of recombinant proteins. In addition, chemical chaperones are found to decrease the toxic effect of IPTG on cells.