Dimerization of core protein is a crucial step in the formation of the hepatitis C virus (HCV) nucleocapsid， and inhibition of dimer formation is regarded as an attractive approach to design anti-HCV drugs. In this work， we developed the atomic force microscopy based single molecular force spectroscopy (AFM-SMFS) method for the characterization of core protein dimerization with the advantages of small amount of sample consumption and no need of labeling. Interaction force of the core protein with its antibody or aptamer was analyzed to investigate its stoichiometry and binding property. The two specific binding forces were detected due to the probing of dimeric and monomeric core protein， respectively. Moreover， the binding property of protein dimer was different from the monomer. Our work offers a new approach to study the dimerization of core protein， as well as other proteins， and to screen the HCV candidate inhibitors.