A novel fluorescent single nucleotide polymorphism (SNP) assay was developed by using Graphene Oxide (GO), which provides a fast, sensitive and simple method for SNP detection. The strategy was based on the single base extension reaction and different absorption capacity of fluorescein labeled dGTP (dGTP-Fl) and double-stranded DNA (dsDNA) to GO. dGTP-Fl is incorporated into the probe by extension reaction for the mutant target but not for the wild target, which leads to recovered fluorescence for the mutant target because of weak interaction between dsDNA and GO and weak fluorescence for the wild target because of the quenched fluorescence of dGTP-Fl by GO. The method shows a linear range for the mutant-type target from 3 nM to 50 nM and 3 nM is the detection limit. It was noted that as low as 10% mutant-type target could be detected in the presence of the wild-type target, in which the concentration is 9 times higher than that of the mutant-type target.