Vibrio parahaemolyticus is a major seafood-borne pathogen causing significant economic losses in aquaculture systems. Therefore, application of phage encoded enzymes, particularly endolysin, as a new strategy for effective biocontrol and therapeutic agent against bacterial diseases. In the present study, we synthesized endolysin gene (Vplys60) of bacteriophage qdv001 and biochemically characterized by expressing in Pichia pastoris X-33. In addition to, we also investigated the anti-biofilm and anti-vibriosis activity of Pichia-expressing Vplys60 against vibrio challenged in vivo aquaculture model, Artemia franciscana. The result indicated that the predicted molecular size of Pichia expressed Vplys60 was approximately 28 kDa as verified by SDS-PAGE and zymogram. Vplys60 manifested stable activity over broad range of pH (6–10), temperatures (37–75 °C) and salinity (100–600 mM NaCl). Biochemical and in silico analysis revealed that addition of calcium ion (Ca2+) enhanced the lytic activity of Vplys60 whereas other metal ions inhibited the activity. Additionally, calcium-dependent Vplys60 has showed a strong amidase activity by cleaving the peptidoglycan of V. parahaemolyticus. Our data also showed that Vplys60 (75 μg/ml) significantly inhibits biofilm formation (91.6%) and significantly reduced the bacterial population. The in vivo challenge study showed enhanced survival rate in combination with reduced vibrio load in Artemia after administration of Pichia-expressing Vplys60. © 2019 Elsevier B.V.