EPSP gene encodes the enzyme of 5-enolpyruvylshikimate-3-phosphate synthase. The enzyme in Allium macrostemon Bunge shows some glyphosate resistance. It may improve the glyphosate resistant ability if transformed into flax (Linum usitatissimum L.). In this study, the gene cDNA (EPSPsA) was formerly cloned from Allium macrostemon and Bunge was inserted into the vector pWM101 to construct a recombinant expression gene. The gene is under the control of a consistent expression promoter 35S. The recombinant gene was transformed into fiber flaxby Agrobacterium tumefacien mediated callus transformation. The transformed calluses are subject to hygromycin screening and resistance callus are induced in induction and differentiation culture. The transformed seedlings are obtained and verified by PCR. The EPSPsA gene was integrated into genome of the flax. The glyphosate resistance detection of transgenic flax callus and seedlings proved that the EPSPsA can improve the glyphosate tolerance of flax.