Vibrio harveyi is a pathogen common in the marine fish culture of China. To explore the pathogenic mechanism of V. harveyi and develop effective methods for prevention and control V. harveyi infection, we extracted extracellular products (ECPs) from the high virulent strain H2LB1 and the low virulent strain H1SAI3 of V. harveyi. The proteins of ECPs were analyzed and identified by using the label-free proteomics method. According to the result of protein identification, we selected the high virulent strain-specific protein HutZ for further analysis. Firstly, qRT-PCR technology was used to detect the expression difference of of V. harveyi gene HutZ under culture conditions with different iron sources. Secondly，the gene HutZ was cloned to the expression vector，and recombinant HutZ protein was successfully expressed and purified. Then the purified recombinant HutZ protein was incubated with hemin, and the spectrum was scanned at 300-700 nm. Finally, Cynoglossus semilaevis were immunized with purified recombinant HutZ protein twice. After immunization, the serum titers of 1-7 weeks were detected by the indirect hemagglutination method, and the challenge experiment was performed after 4 weeks of immunization. The results showed that the expression of gene HutZ is related to the iron source; HutZ protein affected the absorbance of hemin; the antibody titer of C. semilaevis against HutZ protein reached up to 1∶128, and the immune protection rate was as high as 73.3%. These results indicated that HutZ protein is a vital factor for the iron uptake of V. harveyi and potential candidate antigen for developing effective subunit vaccines to prevent the V. harveyi infection.