Gene fusion is a molecular event occurring in cellular proliferation and differentiation, and the occurrence of irregular fusion gene results in various malignant diseases. So, sensing fusion gene with high performance is an important task for integrating individual disease information. Here, we proposed a nonenzymatic and high-throughput fluorescent assay system for the detection of fusion gene by employing DNA nanotweezers with hydrolytic activity. This tweezer was assembled by three single-stranded DNAs and engineered with sensing elements and reporting subunits. In the absence of the fusion gene, the engineered tweezer remained opened and inactive which led to no signal output. However, the addition of fusion genes would cause structure alterations of the tweezer from open to close and further DNAzyme activation with the assembly of two reporting subunits. Then, the activated DNAzyme catalyzed fluorescence substrates for signal conversion. Taking BCR/ABL fusion gene as an example, the tweezer-based assay system showed not only excellent distinguishing capability towards different input targets but also high sensitivity with a detection limit of 5.29 pM. In addition to good detection performance, this system was simple and enzyme-free, offering a powerful nanometer tool as a smart nanodevice for sensing fusion detection.