The invention discloses a composition for modifying a nucleotide sequence, a method and application, and relates to the technical field of gene editing. The composition comprises a first vector and asecond vector, wherein the first vector is provided with the following expression elements, such as a cytosine deaminase expression element, an adenine deaminase expression element and a mutant Cas enzyme expression element; the second vector is provided with the following expression elements, such as a gRNA (guide ribonucleic acid) expression element and a uracil glycosidase inhibitor expressionelement. The composition is used for modifying the C bases at the 1st to 16th sites at the upstream of PAM (protospacer adjacent motif) of the target nucleotide sequence, so that the transformation ofC/G to T/A is realized. Compared with the existing gene editing technique, the composition and the method have the advantages that the working window is broader; the DSB (double-strand break) and indels (insertion/deletion mutations) are not introduced, the off-target effect is very low, the safety is higher, and the like.