The antimicrobial activity and DNA binding mechanism of a novel antimicrobial peptide (AMP) PV13 against Vibrio parahaemolyticus from Penaeus vannamei were explored. The sequence of the peptides were identified by using ultra-high-performance liquid chromatography-high resolution mass spectrometry， and the cationic antimicrobial peptide PV13 (ALPWVLPWALPRALPRVLPR) was screened by using in silico tools. The antimicrobial activity of AMP PV13 against V. parahaemolyticus was evaluated by minimum inhibitory concentration (MIC) and time-kill curve， the MIC of PV13 was 62.5 μg/mL and killed V. parahaemolyticus within 2 h. The antibacterial mechanism of PV13 against V. parahaemolyticus was evaluated using transmission electron microscopy (TEM)， intracellular membrane permeability， DNA gel retardation and circular dichroism. The results shows that PV13 increases bacterial cell membrane permeability and binds bacteria genomic DNA concentration dependently. Moreover， AMP PV13 exhibited a random coil structure in PBS and SDS solutions， while its structure changed significantly after binding with DNA of V. parahaemolyticus. The antimicrobial activity of peptide PV13 could be due to the presence of four leucine-proline repeat regions. The above results provide new insight into screening and designing AMPs from P. vannamei， and offer a theoretical basis of AMP PV13 for their potential applications in food preservation.