Single nucleotide polymorphisms (SNPs) are frequently associated with various gene-related human diseases, whose determination has attracted great interest. Herein, we report a graphene oxide (GO) nanosheets-based fluorometric DNA biosensor to study the type and location of the single-base mismatch, as well as the influence of the length of the strands. The results indicated that both short and long targets led to much lower fluorescence signals than the perfectly complementary target, while the type of mismatched base had negligible influence on the results. Furthermore, targets with mismatch location near the 5' end led to higher fluorescence intensity than those near the 3' end when the dye was tagged at the 5' end of the probe.