Objective To investigate the application of DNA microarray to the screening of deafness gene mutations. Methods Twenty one deaf patients aged from 8 to 18 years were extracted for peripheral blood. DNA microarray was applied to detecting mutations of 9 hotspots in four most common pathologic genes, namely GJB2 (35delG, 176del16, 235delC, 299delAT), GJB3 (C538T), SLC26A4 (IVS72A>G, A2168G) and mitochondrial 12S rRNA (A1555G, C1494T). Results SLC26A4 gene mutations were detected in 8 cases (38%), including IVS72A>G heterozygous mutation in 7 and IVS72A＞G and 2168A＞G heterozygous mutation in one. Four cases (19%) carried GJB2 gene mutations, including 176 del 16 heterozygous mutation (n=1), 299 delAT heterozygous mutation (n=1), 176 del 16 and 235 del C heterozygous mutation (n=1), and 235 del C homozygous mutation (n=1). Conclusion DNA microarray is a sensitive and specific method for screening sequence variation in deafness gene.