Luciferases identified or engineered so far emit violet, blue, blue-green, green, yellow, red or near infra-red light. The unique and beautiful bluish-green bioluminescence of fireworms Odontosyllis spp. has attracted particular interest, however, their molecular basis is totally unknown partly due to the difficulty of animal collection. Here we report a novel type of luciferase gene from the Japanese fireworm O. undecimdonta. The major SDS-PAGE band of the luminous mucus showed luciferase activity. A highly sensitive mass spectrometry analysis in combination with RNA sequencing technique revealed that this band was product of a single gene with no homology to any other sequences in public databases. The recombinant protein of this putative luciferase gene expressed in mammalian cells produced the same unique bluish-green emission peak as the fireworm crude extract, indicating that this novel gene is the genuine fireworm luciferase with an evolutionary different origin from other luciferases previously described. Our findings extend the repertoire of luciferin/luciferase system to previously unavailable wavelength range.