Vibrio parahaemolyticus, a common foodborne pathogen, can form biofilms for survival in various environments and for bacterial transmission. Lux systems in Vibrio species are the typical two-component signal transduction systems, which have been demonstrated to contribute to various phenotypes; however, the functions of each homolog of the Lux system in V. parahaemolyticus in the regulation of biofilm formation remain largely unknown. In this study, we first showed that LuxQ, LuxU, and LuxO are essential for controlling biofilm formation by V. parahaemolyticus, through gene knockout studies. We also found that they acted in the same signaling pathway and their deletion mutants exhibited a similar level of biofilm formation. Furthermore, site-directed mutagenesis revealed that the conserved residues for phosphorylation in LuxQ (D784), LuxU (H56) and LuxO (D47) were critical for their regulatory functions on biofilm formation. Phos-tag™ sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the phosphorylation of LuxU and LuxQ in vivo. Finally, qPCR analysis displayed that the three mutants had a significant decrease in the transcription level of cps loci and cpsQ compared with the wild type strain, which is consistent with the observed phenotype of biofilm formation. Therefore, we propose that LuxQ and its downstream factors LuxU and LuxO function in the same signaling cascade to control biofilm formation by regulating the expression of cpsQ and cps loci. The results of this study provide new data regarding the role of the LuxQ-LuxU-LuxO pathway in biofilm formation by V. parahaemolyticus and help further understand the complex regulatory functions of Lux pathways.