With the development of biotechnology, the detection of cancer biomarkers has been a promising approach for cancer diagnosis and therapy. Herein, we reported a DNA octahedron-based fluorescence nanoprobe, which was capable of detecting and imaging of two kinds of tumor-related mRNAs in living cells simultaneously. The DNA nanoprobe was constructed of eight single-stranded DNAs, in which two oligonucleotides (recognition sequences) were modified with quenchers (BHQ2 and BHQ3) and the adjacent sequences were modified with fluorophores (Cy3 and Cy5), respectively. In the presence of targets, the recognition sequences could dissociate from the nanoprobe skeleton by strand displacement reaction and the fluorescence was recovered accordingly. With the modification of AS1411 aptamer, the nanoprobe could internalize cancer cells more efficiently and distinguish cancer cells from normal cells. In addition, the nanoprobe exhibited good stability, biocompatibility, selectivity, and responded quickly to the targets as well. The DNA nanoprobe was designed in the formation of octahedron and may provide an inspiration for multidetection of cancer biomarkers based on the DNA nanotechnology.