The fireworms Odontosyllis spp. are globally distributed and well-known for their characteristic and fascinating mating behavior, with secreted mucus emitting bluish-green light. However, knowledge about the molecules involved in the light emission are still scarce. The fireworms are believed to emit light with a luciferin-luciferase reaction, but biochemical evidence of the luciferase is established for only one species living in Japan and no information is available for its luciferin structure. In this study, we identified a luciferase gene from a related Puerto Rican fireworm. We identified eight luciferase-like genes in this Puerto Rican fireworm, finding amino acid identities between Japanese and Puerto Rican luciferase-like genes to be less than 60%. We confirmed cross reactivity of extracts of the Japanese fireworm luciferin with a recombinant Puerto Rican luciferase (PR1). The emission spectrum of recombinant PR1 was similar to the crude extract of the native luciferase, suggesting that PR1 is a functional luciferase of this Puerto Rican fireworm. Our results indicate that the molecular mechanism of luminescence is widely conserved among fireworms.