Abstract: Objective: To increase the secretory expression level of the human single chain variable fragment (scFv) of ant-i digoxin antibody(ADA) cloned from a hem-i synthetic phage antibody library. Methods: (1) The ADA scFv gene was amplified from the experssion plasimd pHEN2-ADA scFv with PCR, recombinated into the plasmid p3MH to construct the expression plasmid p3MH-ADA scFv,and transformed into E.coli strain HB2151and XL1-blue. (2) ADA scFv was secreted into the culture medium as soluble protein in E.coli srain HB2151carrying the two expression plasmid mentioned above and E.coli strain XL1-blue carrying expression plasmid p3MH-ADA scFv,respectively. ELISA was used to analyzed the soluble ADA scFv in the supernant of the culture medium. (3) ADA scFv was secreted into culture medium with addition of various concentrations of sucrose, glycine,and Triton X-100in the E.coli strains mentioned above. The soluble ADA scFv in the supernant of the culture medium was analyzed with ELISA. Results: (1) The level of the soluble ADA scFv in culture medium expressed in E.coli srains of HB2151and XL1-blue carrying expression plasmid p3MH-ADA scFv was 5-fold of that expressed in E.coli HB2151 carrying expression plasmid pHEN2-ADA scFv. (2)Addition of sucrose to the medium gave more than5-fold increase in the level of soluble ADA scFv expressed in E.coli HB2151and XL1-blue carrying expression plasmid p3MH-ADA scFv. Conclusions: The level of the soluble ADA scFv in culture medium was increased more than 25-fold when expressed in E.coli srains of HB2151and XL1-blue carrying expression plasmid p3MH-ADA scFv combined with the addition of sucrose to the culture medium.