Flavobacterium psychrophilum (F. psychrophilum) is the causative agent of bacterial cold-water disease (BCWD) that occurs in ayu Plecoglossus altivelis. Formalin-killed cell of F. psychrophilum has long been studied as an immersion vaccine for BCWD. In this study, we explored the possibility of F. psychrophilum collagenase (fpcol) for use as the immersion vaccine. BCWD convalescent ayu sera contained specific IgM antibodies against somatic F. psychrophilum and fpcol, meaning that fpcol is a promising antigen for the vaccine development. The recombinant fpcol was successfully expressed in Escherichia coli and Brevibacillus chosinensis (B. chosinensis). The culture supernatant of the B. chosinensis was used as an immersion vaccine solution. The vaccinated ayu were then challenged by soaking into F. psychrophilum culture. In two experimental groups, the relative percentages of survivals were 63 and 38%, respectively, suggesting that fpcol is promising as the immersion vaccine for ayu-BCWD.