We presented a low-abundance mutation detection method with lambda exonuclease and DNA threeway junction structure.The assistant strand in the DNA three-way junction structure could regulate the reaction system from the kinetics and thermodynamics aspects.The optimization of the assistant strand helps to improve the selectivity of the mutant-type DNA to the wild-type DNA about 35 times.Moreover,the cost of the optimization process could be saved by about 90％.The method was applied to the detection of a human ovarian cancer-related gene mutation BRCA1 (rs1799949,c.2082C＞T).The limit of detection to the mutation abundance in the DNA three-way junction structure system (0.2％) was one order lower compared with that in the double-stranded DNA structure system (2％).The mutation abundance in different standard samples was quantitively measured,and the results were consistent with the initial abundance in the standard samples.