This study aims to isolate the antimicrobial peptide (AMP) from buffalo casein hydrolyzed by Dregea sinensis Hemsl. protease. The AMP was isolated from hydrolysate by live bacteria adsorption, then analyzed using reversed-phase high-performance liquid chromatography, and the fraction with highest antimicrobial activity was identified by liquid chromatography-tandem MS. Further, we characterized the peptide in terms of its peptide sequence, structure, and antimicrobial activity. The results identified the AA sequence of the peptide as YLGYLEQLLRLK, which corresponds to residues 106 to 117 of bovine αS1-casein, and we named it BCp12. BCp12 displays α-helical structure, with high hydrophobic moments and net positive charge. BCp12 can inhibit the growth of indicator bacteria, with minimum inhibitory concentration values ranging from 0.8 to 1.6 mg/mL, and can induce low toxicity in mammalian cells. Antimicrobial activity of the BCp12 peptide remained stable under different salt concentrations but was sensitive to trypsin and high temperatures (121°C and above). The results support further research in the application of our newly generated AMP as an antimicrobial agent in the food industry and in food processing facilities.