Vibrio alginolyticus is one of the main pathogens that causes mass mortality in aquatic animals and infects humans. To reduce the application of antibiotics, alternative therapies have been proposed. One of the promising possibilities is to control diseases through the use of lytic bacteriophages. As few current bacteriophages against V. alginolyticus have been reported, the main objective of this study is to develop effective bacteriophages for controlling pathogenic bacteria. This study describes the isolation and characterization of lytic bacteriophages against a V. alginolyticus strain (VAHN1). The bacteriophages were isolated from Hainan shrimp pond and Fujian marine products by double-layer agar culture method, and V. alginolyticus VAHN1 was used as the host strain for bacteriophage isolation. The phages were classified and identified by transmission electron microscope (TEM), restriction endonuclease and phylogenetic tree analysis.The physiological and biochemical characteristics of the bacteriophages were determined, including optimal multiplicity of infection (MOI), lysis spectrum detection, pH stability, thermal stability, resistance to UV light, and sensitivity to chloroform and ether. In addition, the effect of bacteriostasis were also measured. The results showed that 2 lytic bacteriophages against V. alginolyticus were isolated, named as VAP9 and VAP21. The plaques of VAP9 and VAP21 were neat and transparent, with a diameter of 1.5-2 mm. The nucleotides of the phage VAP9 and VAP21 were all dsDNA, and all their heads were shown as icosahedral shape with about 55 nm and 65 nm diameter respectively under TEM. The tail of the phage VAP9 was approximately 65-70 nm in length and 15 nm in width, while the tail of the phage VAP21 was about 75-80 nm in length and 18 nm in width. The 2 bacteriophages are grouped under the Myoviridae family. The phage VAP9 and VAP21 had good tolerance to different physical and chemical environment. The survival rate of the 2 bacteriophages was greater than 43% at 60 °C for 2 h. The optimal pH of VAP9 was 6-8 and VAP21 was 7-11. The 2 bacteriophages tolerated peracetic acid in universal bactericidal concentration. The phages VAP9 and VAP21 were insensitive to chloroform and ether, with a certain resistance to UV light. The optimal MOIs of both VAP9 and VAP21 were 0.001. The cocktail of VAP9 and VAP21 was able to infect 95.2% (157 strains among 165 strains) of the V. alginolyticus strains and 50% (3 strains among 6 strains) of the V. parahaemolyticus strains used in this study. And they could not infect other species of tested bacteria except V. alginolyticus and V. parahaemolyticus. The phages VAP9 and VAP21 could effectively inhibit the growth of V. alginolyticus VAHN1 and have the same inhibitory trend on VAHN1, but the VAP21 strain had a stronger bacteriostatic effect than that of the VAP9 strain. Moreover, the inhibitory effect involved the cocktail of 2 bacteriophages on V. alginolyticus were better than that of a single phage. Alignment with the sequences of the conserved protein amino acid sequences of the phage VAP9 and VAP21 on National Center for Biotechnology Information (NCBI), it was showed that the 2 bacteriophages had low homology with other bacteriophages. Furthermore, phylogenetic and genome analysis revealed that the phages VAP9 and VAP21 have low homology with other bacteriophages and may be 2 novel Myoviridae bacteriophages that infect bacteria related Vibrio spp. This study did not only enrich the species resources of bacteriophages against V. alginolyticus, but it also laid a theoretical foundation for the development and application of Vibrio bacteriophages as microecological antimicrobial agents.