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Species identification is of key significance for exploring the origin and transmission of ancient silks. In this study, two novel methods, i.e.western blot (WB) and proteomics analyses, were proposed and established to identify the differences between silks from Bombyx mori(B. mori) and two other distinctive species (Eri silkworm and Chestnut silkworm). Three diagnostic antibodies, a polyclonal anti-silk fibroin (anti-SF) antibody (pAb), a polyclonal anti-SF-specific peptide antibody (pAsb), and a monoclonal anti-SF antibody (mAb) were designed and prepared to distinguish silk species using the antibody-based WB technique. Proteomics analysis by liquid chromatography–tandem mass spectrometry was performed to further identify silk species at the protein level. WB results indicated that the three antibodies showed high specificity and affinity and could discern B. morisilk from Eriand Chestnutsilks. Biomarkers for each SF were obtained using proteomics analysis, and they have the potential to serve as standards for identifying silk species. Thus, combining WB and proteomics analyses with conventional methods can provide more accurate silk information and may be suitable for identifying other proteinaceous materials in archaeological field.