Functional genomics require high-throughput mutation detection and S1 nuclease mutation detection is one of them, which has the advantages of low-cost and rapidness.But it is difficult to conquer the reaction and some perfectly matched sequence also will be degraded non-specificly. In this paper, the targeted mutagenesis promoter of chlorophyll a/b binding protein was exemplified, the procedure was streamlined and the cause of degrading duplex DNA by S1 nuclease non-specificly had also been elucidated.