The invention provides a kit for detecting mitochondrial DNA mutation related to a regulon of a Leber disease, wherein the kit consists of primer sequences, reagents for extracting genome DNA of samples, reagents for PCR amplification reaction, a positive control sample, a negative control sample and an instruction, restrictive endonucleases comprise a restrictive endonuclease Nla III, and the positive control sample is a restriction enzyme digested sample in which 4435th site of a mitochondrial sequence occur A to G mutation. The kit provided by the invention can be applied in the detection of mitochondrial DNA mt DNAA4435G mutation related to the regulon of the Leber disease. According to the kit, specificity and stability of detection results can be ensured, cost is lower than that of other detection methods, and the detection process is simple, quick, accurate and economic, and thus is suitable for detecting LHON-related gene mutation.