The plasmid pTE-okm, which was obtained by inserting the fluorescence gene eyfp into the plasposon pTnMod-okm, transferred from E.coli S17-1 into Shewanella decolorationis S12 by conjugation. pTE-okm could not replicate in Shewanella decolorationis S12 because of its narrow-host-range replication origin, but the minitransposon within pTE-okm could transpose to the chromosome of Shewanella decolorationis S12 and made it survive the LB medium with kanamycin and rifampicin. The clones which expressed fluorescence gene eyfp were screened from all the clones on the selected plates under fluorescent microscope and it was make sure that pTE-okm didn't exist in these clones. S12-40, which had the the same growth rate and decoloration ability with the original strain S12-1, was picked out within these clones. S12-40 expressed EYFP stably even after successive re-culturing for 20 times (8h/time). The construction of S12-40 laid a foundation for the study on the ecological performance of Shewanella decolorationis S12.