Vibrio alginolyticus, an opportunistic pathogen in humans, obtains iron from various iron-containing compounds, including hemin, a heme derivative. This process may be an important virulence-associated adaptation for evolution into a human pathogen. The easiest way for a pathogen to acquire iron from a host is through erythrocyte lysis by hemolysins, which increases the available iron pool in vivo. Hemolysins are important virulence factors in many bacterial pathogens. In this study, a DNA fragment containing the 1281 bp ORF of the hemolysin gene from V. alginolyticus was identified by isolating a hemolytic-negative mutant from a random mutagenesis library. The external DNA sequence from the transposon inserted during random mutagenesis was identified by DNA sequencing. The hemolysin gene was amplified and cloned into the expression vector pET28a. The expression vector containing the hemolysin gene was transformed into Escherichia coli BL21(DE3), and the overexpressed protein had approximately 47 kDa molecular weight, as predicted by SDS-PAGE. The hemolytic activity of whole-cell lysate with overexpressed protein AGV17462.1 was tested, and it shows hemolysis slightly less than the positive control, which means 100% hemolysis. No hemolysis was observed after heat treatment at 100 °C for 10 min. Therefore, it is considered a thermolabile hemolysin. In conclusion, transposon-induced gene disruption leads to the loss of the hemolytic phenotype in the isolated mutant, in which the DNA sequence has been identified and characterized as a new hemolysin gene.