Objective：This study aims to identify the molecular etiology of two non-syndromic hearing loss families，and to use zebrafish to analyze the function of WFS1 on the occurrence of hearing loss. Methods：By using targeted capture sequencing technology，exon sequencing analysis was performed on two families to identify candidate pathogenic genes. The function of human WFS1 gene and the homology of WFS1 gene between zebrafish and human were analyzed by bioinformatics. The temporal and spatial expression characteristics of zebrafish wfs1a and wfs1b were analyzed by whole-embryo in situ hybridization and quantitative PCR. Results：Exon sequencing and co-segregation analysis of two families suggested that the heterozygous mutation of the WFS1 c.2036~2038delAGG（p.680delE） and c.1957C>T（p.653R> c）was the molecular pathogenesis basis of the pedigree JSNY-021 and JSNY-033，respectively. Quantitative PCR and whole-embryo in situ hybridization results showed that zebrafish wfs1a and wfs1b showed different spatio-temporal expression characteristics at different embryonic development stages. Bioinformatics analysis suggested that wfs1b had a closer evolutionary distance and higher homology with human WFS1. Conclusion：This study confirmed that the molecular etiology of two autosomal dominant non-syndrome hearing loss families are WFS1 mutations，which expanded the gene mutation spectrum of hereditary hearing loss. The expression of wfs1a/wfs1b in the embryonic development of the zebrafish model has obvious spatio-temporal specificity. Wfs1b is the direct homologous gene of human WFS1. The results of this study not only provide support for the molecular diagnosis of hearing loss，but also lay a foundation for further study on the mutagenesis mechanism of WFS1.