C57BL/6Cya mice have two similar adult β-globin protein-encoding genes, Hbb-bs and Hbb-bt. These two genes are located at adjacent positions on mouse chromosome 7 and contain three exons. Hbb-bs & Hbb-bt DKO mice are a β-thalassemia disease model constructed by simultaneously knocking out the Hbb-bs gene and Hbb-bt gene in C57BL/6JCya mice using gene editing technology. This model is homozygous lethal, and heterozygous mice show typical features of severe thalassemia, such as abnormal hemoglobin content, red blood cell count, hematocrit, mean corpuscular hemoglobin concentration, red cell distribution width, platelet count, spleen size, and red cell morphology, and have reproductive ability.