The invention belongs to the technical field of gene detection and particularly relates to a kit for detecting mutation of deafness susceptibility genes and an application of the kit. A PCR mixture in the kit contains 17 primer single strands shown as SEQ ID NO: 1-17 and 11 probes shown as SEQ ID NO: 19-29, genotypes of 9 mutation sites of four deafness susceptibility genes and one reference gene can be detected simultaneously in a single-tube PCR system, the genotype of a sample is acquired through fluorescent PCR melting curve analysis after PCR amplification is ended, the whole operation can be finished within 2-3 h, a few operation steps are adopted, short time is consumed, many single-tube detection sites are adopted, and the flux and mutation coverage are high; besides, the kit adopts homogeneous-phase detection and closed-tube operation, so that pollution of a PCR product is reduced, detection specificity is high, and a result is easy to interpret.