Background: Due to extreme genetic heterogeneity, targeted next-generation sequencing (NGS) can be an efficient tool for rapid genetic diagnosis of hereditary non-syndromic hearing loss (NSHL). Aims/Objectives: This study was aiming to identify causative variants in NSHL patients from the Minnan region through targeted NGS. Material and methods: Genomic DNA samples from 90 NSHL subjects were extracted and subjected to SureSelect target enrichment system to capture the entire coding exons and flanking intronic regions of gene GJB2, SLC26A4, and MT-RNR1. The captured DNA was then sequenced by Illumina HiSeq2500. The sequence data was processed and quality-checked using Burrows-Wheeler Alignment, Picard, and GATK, and annotated by SnpEff, SIFT, and GERP++. Results: Twenty-six candidate variants in GJB2, SLC26A4, and MT-RNR1 were detected in 57 of 90 NSHL patients. GJB2 c.109G > A was the most frequent variant, followed by GJB2 c.608T > C and c.235delC. Genetic diagnosis was available for 22 NSHL patients, including 19 patients associated with autosomal recessive NSHL, one patients with autosomal dominant NSHL, and two individuals with mitochondrial disorders. Conclusions and significance: Our targeted NGS analysis added supports for the application of NGS in routine diagnosis and provided an overview of genetic variants with allele frequencies in the deaf population from the Minnan region.