<p>Background: CD20 is an important cell surface receptor that is used for target therapy of B cell lymphoma and some related blood diseases due to vital function of CD20. In previous studies, a Rituximab based humanized single chain variable fragment (scFv) antibody showed good reactivity against B cell related cancer cells. But this recombinant protein produced Inclusion Bodies (IBs) in <em>Escherichia coli (E. coli)</em> cytoplasm. The aim of this study was to investigate the effect of coexpression with cytoplasmic chaperones on expression and solubility of humanized anti-CD20 scFv in <em>E. coli</em>.<br /> Methods: For this purpose, the fragment coding for anti-CD20 huscFv subcloned into the pET22b (+) and transformed into the <em>E. coli</em> BL21 (DE3) was evaluated. In order to inhibit the production of IBs, the effects of co-expression with cytoplasmic chaperones GroEL, DnaK, GroES, Tig, DnaJ and GrpE were investigated.&nbsp;<br /> Result: Coexpression with cytoplasmic chaperones led to increased soluble expression of anti-CD20 recombinant protein. Among investigated chaperones, pKJE7 chaperone plasmid containing DnaJ, GrpE, DnaK chaperone genes had significant effects with an expression yield of 325 <em>&micro;g/ml</em> soluble anti-CD20 scFv.&nbsp;<br /> Conclusion: The result of this study demonstrated remarkable effect of pKJE7 chaperone on enhancement of soluble expression of anti-CD20 huscFv antibody in <em>E. coli</em>.</p>