Shorten the length of DNA strand weakens the duplex stability,leading transiently binding between complementary sequences[1,2].The binding kinetics is highly dependent on the number of base pairs[3].Herein,we developed a single molecule assay based on DNA transient binding for single nucleotide variants detection on total internal reflection fluorescence microscopy.Statistical characterization of the single molecule fluorescence trajectories enabled us absolutely discriminating wild type and single nucleotide variants at single molecule level.The model mutant(KRAS c.34A)can be clearly differentiated from the wild type(KRAS c.34G)at an abundance as low as 0.01%.The results of two cancer cell lines having high incident KRAS and BRAF mutation were tested showing the feasibility of the method for biological and clinical samples.