Klenow fragment(KF) uses the activity of a separate exonuclease to excise nucleotide,which is a crucial step in DNA replication and repair.Here is a novel sensitive and convenient method introduced for real-time monitoring nucleotide excision by KF with a molecular beacon as a detecting probe in a homogeneous solution.This method,which overcomes the drawbacks of traditional methods such as discontinuity,time consuming and low sensitivity,was used to assay KF activity and the detection limit reached up to 0.4 U/mL.In addition,the method was applied to investigating the effects of metal ions and chemical drugs on the reaction.The results demonstrate that it is a potential high-throughput assay for screening inhibitors and activity analysis of KF in vitro.