The invention provides a western blot kit for detecting the antibody of autoimmune disease and a preparation method of the western blot kit, and relates to a western blot kit for detecting related antibodies of various autoimmune diseases, aiming at overcoming the technical defect that a western blot product is unavailable for testing and screening various autoimmune diseases in the prior art. The nitrocellulose membrane or the nylon membrane contains at least two parallel detection lines coated by at least two of ten natural antigens or recombinant antigens, i.e. dsDNA (deoxyribonucleic acid), Sm / RNP (ribonucleoprotein), CCP (critical compression pressure), SSA (sulfosalicylic acid), SSB (single-strand binding protein), GAD (glutamic acid decarboxylase), ICA (islet cell antibody), IA-2A (islet cell), TG (triglyceride) and AMA-M2 (anti-mitochondrial antibody), a high-concentration quality control band, a median-concentration quality control band and a low-concentration quality control band. The deficiency of the detection sensitivity and the specificity of the single autoantibody can be overcome, the operating complexity for independently detecting the related autoantibody of various diseases one by one can be overcome, and the detection efficiency and the result judging accuracy degree can be greatly improved.