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<i>Aeromonas veronii</i> is a virulent fish pathogen that causes extensive economic losses in the aquaculture industry worldwide. In this study, a virulent strain of <i>A. veronii</i> TH0426 was used to establish an in vitro biofilm model. The results show that the biofilm-forming abilities of <i>A. veronii</i> TH0426 were similar in different media, peaking under conditions of 20 &#176;C and pH 6. Further, isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics methods were used to compare the differential expression of <i>A. veronii</i> between the biofilm and planktonic cells. The results show alterations in 277 proteins, with 130 being upregulated and 147 downregulated. Pathway analysis and GO (Gene Ontology) annotations indicated that these proteins are mainly involved in metabolic pathways and the biosynthesis of secondary metabolites and antibiotics. These proteins are the main factors affecting the adaptability of <i>A. veronii</i> to its external environment. MRM (multiple reaction 27 monitoring) and qPCR (qPCR) were used to verify the differential proteins of the selected <i>A. veronii</i>. This is the first report on the biofilm and planktonic cells of <i>A. veronii</i>, thus contributing to studying the infection and pathogenesis of <i>A. veronii.</i>