Autosomal dominant non-syndromic hearing loss (ADNSHL) caused by MYO6 gene variation typically manifests as progressive post-lingual deafness. We identified an 11-member Chinese family across four generations with ADNSHL. We investigated the genetic patterns and mechanisms and employed whole-exome sequencing on genomic DNA derived from peripheral blood to screen and identify pathogenic genes. We verified the potential pathogenicity of the mutation site through minigene assays. Our investigation revealed a novel splice variant, c.554-4A > G (NM_004999.4), located in intron 7 of MYO6, which exhibited clear co-segregation with the hearing impairment phenotype among the family members. Subsequent minigene splicing assays demonstrated that the c.554-4A > G variation resulted in an insertion of three bp in intron 7 of MYO6 (c.554-1_554-3 insTAG, p.184_185insV). The novel splice variant: c.554-4A > G was associated with ADNSHL. Our study expands the spectrum of pathogenic genes and variant sites associated with ADNSHL.