The invention discloses a kit used for detecting the relevant ANA spectrum of self-immune disease, and a preparation method thereof. The kit comprises a reaction card, an enzyme-linked reagent, a developer, a stopping solution and a concentrated washing solution; the reaction card consists of a carrier sheet and a nitrocellulose film or a nylon film which is fixed on the carrier sheet; the nitrocellulose film or the nylon film comprises 15 parallel detection lines which are wrapped respectively by nRNP / Sm, Sm, SS-A, Ro-52, SS-B, Scl-70, Jo-1, nucleosome, dsDNA, histone, ribosome P protein, AMA-M2, PM-Scl, CENP B, PCNA15 natural and recombinant antigen. The kit of the invention can exactly diagnose the self-immune disease, overcomes the shortages of the existing immunoblot diagnosis methodon detecting the self-immune disease, and has the advantages of high sensitiveness, strong specificity, good exactness, simple and convenient operation, little consumption of the samples, and objective result judging, etc.