Glyphosate and glufosinate are two widely used non-selective herbicides globally. The former inhibits the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the plant's shikimate acid metabolic pathway, while the latter inhibits the glutamine synthetase (GS) essential for plant ammonia assimilation. Both herbicides can non-selectively kill annual plants, including crops. The introduction of glyphosate-resistant genes into crops confers herbicide tolerance (HT) to the crops, which holds significant value in the field of molecular breeding. This study isolated a halophilic bacterium (Halomonas sp.) with high resistance to glyphosate from marine bacteria. Through genome sequencing and bioinformatic analysis, the coding gene (fHoEPSPS) for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in this strain was identified to encode a bifunctional enzyme (fHoEPSPS) fused with a prephenate dehydratase (PDT) domain. The full-length fHoEPSPS, the G384A mutant (mfHoEPSPS), and a mutant lacking the N-terminal PDT domain (mHoEPSPS) were recombinantly expressed and purified in E. coli (DE3). Glyphosate resistance analysis revealed that the resistance of mfHoEPSPS was 19 times higher than that of fHoEPSPS. The introduction of the mHoEPSPS coding gene into tobacco confers a 3 times the recommended dosage of glyphosate tolerance. Moreover, to develop enzyme with both glyphosate and glufosinate tolerance, we constructd a chimeric enzyme by linking the glufosinate -resistant enzyme (Repat) at the N-terminus of mHoEPSPS with the self-cleaving peptide LP4/2A, resulting RLH. Tobacco transformed with the RLH gene exhibited simultaneous tolerance to 3-5 times the recommended dosage of glyphosate/glufosinate compound herbicides. This research provides new genetic resources for the breeding of HT crops and provide us new insights into the cultivation of dual-resistant glyphosate/glufosinate crops to address the evolution of glyphosate-resistant weeds.