Abstract: Application of a fragment of an isolated nucleotide sequence in the construction of zebrafish without intermuscular bones. The nucleotide sequence is shown in SEQ ID NO:1. Gene mutation is performed by taking SEQ ID NO:1 as a target gene; the mutant F0 embryos are selected and cultured to adult fish; F0 mutant is hybridized with wild type zebrafish to generate an F1 embryos; sense mutant heterozygotes F1 is screened out and cultured to adult fish; and then F1 heterozygote self-crosses to generate F2 generation of three gene types, including homozygote, heterozygote, and wild type. Zebrafish without intermuscular bones is obtained by using a gene mutation method, which provided a basis for subsequent research on a molecular formation mechanism of fish intermuscular bones and the cultivation of economic fishes without intermuscular bone and possessed a basic research value and an application value in other economic aquaculture fish species.