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[Objective] The present study aimed to reveal the relationship between the amino acid sequence, secondary structure, tertiary structure and their functions of the three keratinases produced by the Streptomyces sp. DJ strain. [Method] Based on whole genome sequencing results of DJ strain, the sequence and structure of its three keratinases were analyzed and predicted by using software, such as Bio-edit, DNAMAN, Discovery Studio2.5 et al, and website like https://swissmodel.expasy.org/,and so on. [Result] DJ strains secreted three kinds of keratinases（K1,K2 and K3）,and the keratinases belonged to serine proteases of the S8 family, and their signal peptides, leading peptides and mature peptides were more consistent in the composition, polarity and length of amino acid residues. Three keratinases were modeled using 5 WSL.1.A as template by the homology modeling technique. The 3-D structure of each keratinase contained six α-helix, two 310-helix and fifteen β-folded secondary structures.K1 contained two disulfide bonds, 1 Ca and 2 Ca sites, while K2 and K3 had only one disulfide bond and 1 Ca site. The Pro270 residues of all three keratinases were respectively located in the loop ring, while only Pro242 residue of K2 was also located in the loop ring. The substrates binding regions of the three keratinases were mainly composed of hydrophobic residues, so these keratinases preferred substrate with strong hydrophobicity. For the substrate binding regions of the keratinases, the S1 and S2 played a key role in substrate specificity. And the substrate specificity was different due to the non-conservative nature of the residues at important sites in the S1 and S2 of the three keratinases. [Conclusion] The differences in amino acid, disulfide bond, proline and calcium binding sites leaded to the different stability of the three keratinases, besides the surface charge distribution and substrate binding regions resulted the complementarity to the feather hydrolysis sites. This might be an important reason for the efficient degradation of feathers by DJ strains. [ABSTRACT FROM AUTHOR]