The present invention discloses a method for obtaining glyphosate-resistant rice by a site-directed nucleotide substitution, and also relates to a method capable of generating a site-directed nucleotide substitution and a fragment substitution. The method for obtaining a glyphosate-resistant plant provided by the present invention comprises the following steps: only substituting threonine (T) at position 8 of the amino acid sequence of a conserved region of endogenous EPSPS protein of a target plant with isoleucine (I), and substituting proline (P) at position 12 with serine (S) to obtain a plant, i.e., a glyphosate-resistant plant. The method provided by the present invention is of great significance in breeding new herbicide-resistant plant varieties. The present invention also discloses a method for utilizing a CRISPR-mediated NHEJ pathway to substitute a region between two gRNA sites by designing the two gRNA sites, thereby realizing a site-directed mutation of a target nucleotide and site-directed substitution of a fragment.