Oxidized LDL (OxLDL) is implicated in endothelial dysfunction as well as the formation and progression of atherosclerosis. It has become evident that the atherogenic properties induced by OxLDL are mainly mediated via lectin-like OxLDL receptor-1 (LOX-1). Over the past decade, much research has been performed to investigate lipid metabolism and atherogenesis using genetically engineered mice. To understand the significance of OxLDL, methods to measure the levels of OxLDL in these experimental animals should be established. Utilizing a chicken monoclonal antibody technique, here, we generated anti-human ApoB antibodies that are able to recognize mouse VLDL/LDL. These antibodies were selected from single chain fragment of variable region (scFv) phage library constructed from chickens immunized with human LDL. One of these antibodies, HUC20, was reconstructed into IgY form. Immunohistochemical analysis revealed that this novel antibody specifically stains atherosclerotic lesions of ApoE-deficient mice, associated with Oil red O positive and macrophage-antigen-positive regions. Furthermore, in combination with recombinant LOX-1, a sandwich enzyme immunoassay was developed to measure the levels of LOX-1 ligands in mouse plasma. The sandwich enzyme immunoassay revealed a dramatic increase in the level of LOX-1 ligands in the plasma of ApoE-deficient mice fed high-fat diet, suggesting a link between the level of LOX-1-ligands and the progression of atherosclerosis in mice. Hence, the chicken anti-ApoB monoclonal antibody HUC20 developed here, could be a useful tool to analyze the role of ApoB-containing lipoprotein in atherogenesis in mice.