A highly glyphosate resistant strain of Halomonas sp. was screened from marine bacteria to breed crops with high glyphosate resistance to cope with the evolution of glyphosate weeds. The gene encoding EPSPS in this strain was identified through sequencing genome and bioinformatics analysis. The fHoEPSPS， mfHoEPSPS （G384A site mutant）， and mHoEPSPS （a mutant with N-end PDT deletion of mfHoEPSPS） were recombinantly expressed and purified in E. coli （DE3）. A gene pyramiding strategy mediated by the self-cleaving peptide LP4/2A was used to locate the glufosinate-resistant enzyme （Repat） at the N-end of mHoEPSPS. A dual resistance to glyphosate/ammonium phosphatase （RLH） was constructed. Tobacco transformed with the RLH gene exhibited simultaneous resistance to glyphosate/glufosinate compound herbicides. The results showed that the EPSPS gene （fHoEPSPS） of this strain encoded an N-end fused with a bifunctional enzyme of prephenate dehydratase （PDT）. The results of analyzing glyphosate resistance showed that the resistance of mfHoEPSPS was 19 times higher than that of fHoEPSPS. Introducing the coding gene of mHoEPSPS into tobacco endowed tobacco with three times the recommended dose of glyphosate tolerance. Tobacco plants transformed with the RLH gene had simultaneous tolerance 3-5 times the recommended dosage of glyphosate/ammonium phosphine compound herbicides. It is indicated that the glyphosate-resistant EPSPS derived from Halomonas sp. is a new type of glyphosate-tolerant enzyme. The enzyme activity is further improved through the G384A site mutation. The transgenic tobacco with RLH gene obtained through gene pyramiding strategy mediated by the self-cleaving peptides shows high glyphosate/glufosinate compound resistance. It will provide ideas for dealing with the evolution of glyphosate weeds.