Objective To prepare a mouse monoclonal antibody against human asialoglycoprotein receptor (ASGPR), and to apply it for detecting ASGPR expression in cell lines and tissues. Methods The structure of ASGPR H1 major subunit was analyzed and the full length of ASGPR1 was selected to synthesize immunizing peptide. cDNA was amplified by RT-PCR and then subcloned into prokaryotic vector pGEX-4T-1. The recombinant protein was expressed by E. coli BL21 and purified for subsequent immunization. The conventional hybridoma technique was used to generate mouse monoclonal antibody. The isotype and the titer were regularly tested. Inhibition experiment was conducted to identify the specific binding of the antibody to ASGPR. Finally, the expression of ASGPR was detected in various intra-hepatic and extra-hepatic cell lines by flow cytometry and in different liver tissues by immunohistochemistry method. Results Monoclonal antibody against human ASGPR was successfully prepared and was identified as IgG1, with the titer reaching 112 800. Inhibition experiment indicated a satisfactory specific binding of the antibody to ASGPR and that the recognition epitope was located in the extracellular domain of ASGPR. Flow cytometric analysis showed various levels of ASGPR expression in intra-hepatic cell lines, but not in extra-hepatic cell lines. Immunohistochemistry detection showed that ASGPR was specifically expressed in the normal liver tissues and hepatocellular carcinoma (HCC) tissues, and the expression in HCC tissues was associated with the differentiation degree, with the expression being significantly higher in well-differentiated HCC than that in the poorly-differentiated HCC (75.0% vs 28.6%, P<0.05). Conclusion We have successfully prepared the monoclonal antibody against human ASGPR with high specificity; the antibody can be used for flow cytometric analysis and immunohistochemistry detection of ASGPR and for clinical distinguish of primary or metastatic liver cancer.