Provided are a K85 mutation-containing plant EPSPS mutant, and an encoding gene and an application thereof. The site at position 85, corresponding to escherichia coli EPSPS, of the amino acid sequence of the plant EPSPS mutant has a K85T or K85I mutation. The mutation of the site increases the resistance of different EPSPS mutants of various plants to glyphosate, and maintains the biological enzyme catalysis activity. A plant or recombinant strain transformed with the plant EPSPS mutant can grow normally in the presence of glyphosate. The plant EPSPS mutant may be used for genetically modified crop cultivation.
