Objectives The multidrug resistant bacteria from canine skin were screened. The derivative peptide based on canine β-defensin-3 (cBD3) was designed and its antibacterial activity was studied. Methods The clinically isolated strains were screened with K-B drug-sensitive disk method. Subsequently, the amino acid sequence of β-defensin-3 (cBD3) was used as a template to design the sequence with amino acid substitution. The optimized peptide cBD3-ABU was selected for chemical synthesis. The antimicrobial activity of cBD3-ABU against multidrug resistant bacteria was detected with broth microdilution method. Results 8 strains of multidrug resistant bacteria from canine skin were obtained through screening. CBD3-ABU with a purity of 95.61% was obtained through solid-phase synthesis. The minimum inhibitory concentration (MIC) of cBD3 against Escherichia coli, S.epidermidis, S.pseudintermedius, and S.aureus was 32, 64, 64, 128 μg/mL, respectively. The antibacterial rate of high concentration cBD3-ABU against Escherichia coli, Staphylococcus epidermidis, Staphylococcus pseudointermedius and Staphylococcusaureus from canine skin was 96.02%, 81.85%, 87.25%, and 98.91%, respectively. Conclusions CBD3-ABU has good antibacterial activity in vitro and has the potential to be further developed into therapeutic drugs.