The invention discloses a Chinese population deaf gene screening kit and an application thereof. The kit comprises PCR (polymerase chain reaction) amplification primers and extension primers as well as a PCR reaction agent, wherein the PCR amplification primers and extension primers are designed by taking mutation of lotus 35, lotus 109, lotuses 176-199, lotus 235, lotuses 299-300 of a GJB2 gene, lotus 1174, lotus 1226, lotus 1229, lotus 1975, lotus 2027, lotus 2162, lotus 2168 and lotus IVS7-2 of an SLC26A4 gene, lotus 1494, lotus 1555, lotus 3243 and lotuses 7444 of a mitochondrial DNA (deoxyribonucleic acid) gene as a detection object; and sequences of the primers are as shown in SEQ ID No.1-SEQ ID No.33; and the PCR reaction reagent comprises polymerase and buffer liquor. The kit disclosed by the invention can be utilized to obtain genotypes of 17 lotuses by one step through simple operation, so that cost is low; and accuracy of detection flux and detection results is greatly improved in comparison with that in the prior art, and therefore, the kit has very good clinical and large-scale application value.