Bax, a key tumor suppressor gene, will not be expressed correctly as a result of single nucleotide mutation in its microsatellite region; Instead, Bax2, an isoform of Bax, is often produced. In addition, lack of the exon 2 due to an alternative splicing, Bax2 has the same sequence as Bax except single base deletion from eight continuous guanines (G8) to G7. Most of the currently available methods for Bax2 detection are inefficient and time-consuming, and/or require the use of labels or dyes. In this work, we reported a label-free nanopore sensing strategy to differentiate between Bax and Bax2 with a DNA polymer as a molecular probe based on alternative spliced sequences. Two DNA molecules were designed to selectively detect Bax and Bax2, respectively. The method was rapid, accurate, and highly sensitive: picomolar concentrations of target nucleic acids could be detected in minutes. Our developed simple and fast nanopore-based detection strategy is not only useful for distinguishing between Bax and Bax2, but also provides a useful tool for detection of other single-base mutations in genetic diagnosis.