The aim of this research is to exploit the expression strategies for two genes encoding a rodent and its humanized version single-chain fragments(scFv) specific for hepatocellular carcinoma (HCC) and compare these two scFves in antigen binding activity. Three vectors were used to express these two genes in different routes of fusion pathway, secrete pathway and intracellular pathway. The refolded single-chain antibodies were examined by antigen capture ELISA. Results showed that inclusion bodies were produced in all of the applied vectors despite of variation of IPTG concentration and culture temperature. Cell ELISA and binding competition inhibition indicated that the humanized single-chain Fv retained the similar affinity as its rodent counterpart. These results implied that the solubility of genetic antibodies are determined primarily by its amino acids sequence; The adopted humanization strategy in previous design made little effect on the natural conformation of complementarity-determining regions(CDR) of the parent antibody. The humanized HCC specific scFv can be further evaluated and developed as therapeutics.