Abstract: Vibrio parahaemolyticus is considered to be one of the threatening pathogens for marine animals, which caused huge annual economic losses to the aquaculture. Biofilm formation, a bacterial defense strategy against environmental threats, contribute to the colonization and survival of V. parahaemolyticus during infection of host. Recent study reported that a gene cluster called scv (syp-like locus in V. parahaemolyticus) was found to be involved into V. parahaemolyticus biofilm production, however, the role of most genes in scv gene cluster, including scvL remains to be elucidated. In this study, V. parahaemolyticus strain SHY1833 was isolated from diseased Pacific white leg shrimp (Litopenaeus vannamei) that exhibited acute hepatopancreatic necrosis disease (AHPND). In order to explore the function of scvL in biofilm formation, the in-frame deletion mutant (ΔscvL) was constructed using the allelic exchange method. Additionally, a complemented strain (ΔscvL: pscvL) and an over-expressed strain (WT: pscvL) were generated by introducing pBBR-scvL into the ΔscvL mutant and the wild-type SHY1833 (WT), respectively. Meanwhile, the cpsA deleted mutant (ΔcpsA) was also made as a control for affecting biofilm formation. Crystal violet staining and SYTO-9 fluorescence assays showed that the scvL deletion significantly increased biofilm formation, while scvL overexpression decreased it. As expected, cpsA deletion resulted in decreased biofilm formation. Therefore, it can be concluded that scvL negatively affected the biofilm formation in strain SHY1833 of V. parahaemolyticus. Additionally, the colony morphology was also examined on Congo red plate, revealing that the colonies of ΔscvL were more opaque and rugose compared with that of WT strain. qPCR analysis showed that the transcriptional level of a number of cps genes was considerably elevated in the scvL deletion strain and downregulated in the scvL-overexpressed strain when compared to the WT strain. Consistently, phenol-sulfuric acid methods for determination of expolysaccharide showed that the deletion of scvL caused the significant increase in extracellular insoluble polysaccharides. In summary, these results offer a basis for clarifying how the scv gene cluster contributes to V. parahaemolyticus biofilm development.