The invention provides a reagent kit for simultaneously detecting multiple deafness genes on a single cell level. The invention also provides a method for simultaneously detecting various deafness gene sites on a single cell level. By adopting a single-cell whole genome amplification technology, a sample type with little content of DNA such as a single-cell sample is effectively amplified to whole-genome DNA with total amount of micro-gram by virtue of MDA (multiple displacement amplification). The obtained DNA is amplified by virtue of PCR of a specific primer, Sanger sequencing is carried out for an amplified product on a sequencing instrument, and various genetic deafness genes can be effectively detected.