Abstract: Object By analyzing the audiological and genetic characteristics of a family of Waardenburg Syndrome(WS), to identify the causative gene. Methods The family of deafness with Waardenburg syndrome was investigated with questionnaires, audiological tests and whole body examinations, and the genetic map of the family with deafness was drawn to analyze its audiological and genetic characteristics. Sanger sequencing technology and the EVA and Waardenburg gene diagnostic kits developed by exon capture combined with next-generation sequencing technology were used for candidate gene identification. Results The family has a total of three generations, five people have undergone audiological testing, three patients have hair depigmentation, blue iris, widen inner canth and broad high nasal root, including 1person with hearing loss, 1person with congenital deafness. The use of Sanger sequencing technology for common candidate gene identification, EVA, Waardenburg gene diagnostic kit screening( developed by Xiangya Hospital of Central South University using exon capture combined with next-generation sequencing technology), found two mutation in the pathogenic gene PAX3, PAX3 NM_181457: exon6:c.803G>T and exon6:c.801delT. The PAX3 gene plays an important role in embryonic development as a member of the PAX family of transcription factors. The c.803G>T and c.801delT mutations on exon 6 of the PAX3 gene, c.803G>T mutation can cause the 268th codon encoded by the PAX3 gene to change from serine to isoleucine. c.801delT mutation can cause the 267th codon to change from phenylalanine to leucine, and the subsequent base sequence is confused, resulting in the early termination of the protein at the 283rd codon, which may seriously affect the structure and function of the gene encoding protein and then lead to disease. After searching mutation database and literature search, the mutation has not been reported internationally. Conclusion Because mutations in the PAX3 gene were detected in both the mother and the proband of this family, the family is an autosomal dominant Waardenburg syndrome family due to PAX3 NM_181457: exon6:c.803G>T and exon6:c.801delT mutations. This study enriched the mutation profile of PAX3 gene and provided a reference for clinical molecular diagnosis and genetic counseling.