Abstract: Two eluting peaks including antimicrobial peptide component were isolated from trypsin zymolyte of yak blood, using the membrane binding property of antimicrobial peptides with bacteria cell, with Sephadex G-15 column, under conditions of 10 mmol/L phosphate buffer as eluent and flow rate 0.4 mL/min. The results of binding assay showed that the two novel antimicrobial peptides YakB-1 and YakB-2 were successfully isolated, combining the repeated purification with reverse phase high-performance liquid chromatography followed by antimicrobial activities assay. Antimicrobial activities indicated that both peptides clearly displayed a very impressive broad spectrum of activities against bacterial and fungus tested. Furthermore, the antimicrobial activities of YakB-2 were more powerful than that of YakB-1 against Gram-positive and Gram-negative bacterial. However, YakB-1 showed stronger antimicrobial activities against fungus than YakB-2.The results provide references for the development and utilization of yak blood resource.