To obtain scFv (single-chain variable fragment) against human ASGPR (asialoglycoprotein receptor), a human non-immune phage antibody library was screened with the recombinant CRD (carbohydrate recognition domain) of rCRDH1 (the H1 subunit of human ASGPR). Anti-rCRDH1 phage clones were obtained after four rounds of screening with rCRDH1-coated immunotubes and single positive colonies were further selected with the expressed thioredoxin–His-S tag of the wild-type pET32c. Two specific anti-rCRDH1 phage clones (named C1 and C2) were transfected intoEscherichia coliHB2151 and induced for secreted expression of scFv antibody. The purified anti-rCRDH1 C1 and C2 single-chain antibodies were characterized by immunoblotting and immunohistochemistry. Both antibodies were found to specifically recognize denatured and native forms of the ASGPR and thus could potentially be used as targeting molecules for gene therapy of hepatocellular carcinoma or other liver diseases.