A new strategy for label-free and sensitive detection of single-nucleotide polymorphism (SNP) based on ligation-rolling circle amplification (L-RCA) and intercalating of methylene blue is developed in the present work. A circular template generated by ligation upon the recognition of a point mutation on DNA targets was amplified isothermally by the Phi29 DNA polymerase. The elongation products were hybridized with the capture probe immobilized on the gold electrode. Methylene blue was used as the intercalator to indicate the mutation occurrence. Making use of the high amplification efficiency of Phi29 DNA polymerase and the remarkable precision of Escherichia coli DNA ligase in differentiating mismatched bases at the ligation site, as low as 40 amol mutated strands can be detected, and the positive mutation detection was achieved with a wild-type to mutant ratio of 5000:1, indicating high sensitivity and high fidelity. Furthermore, the proposed sensor is label-free and easy to regenerate compared with most of RCA strategies which utilized an immobilized primer and a labeled detection probe, making it a promising candidate for SNP genotyping.