DNA single-strand breaks (SSBs) have attracted much interest since they are highly related to carcinogenesis and ageing. Herein, we report a new liquid crystal (LC)-based sensor for the detection of DNA SSBs generated by reactive oxygen species (ROS) created from the Fenton reaction. The adsorption of single-stranded DNA (ssDNA) onto the cationic surfactant-laden aqueous/LC interface interferes with the surfactant layer, inducing a planar orientation of the LCs. However, the DNA SSBs generated by the Fenton reaction-produced ROS result in a decrease of the electrostatic interactions between the ssDNA and cationic surfactant molecules, causing rearrangement of the surfactant layer and reorientation of the LCs back to a homeotropic alignment. The changes in orientation of the LCs caused by the DNA SSBs are simply converted and observed as a shift from a bright optical image of the LCs to a dark one under a polarized light microscope. With this simple LC-based approach, the DNA SSBs could be detected more effectively and rapidly without any complex instrumentation or intricate processes. Therefore, our research provides a novel strategy for the detection of DNA damage as well as better insight into the DNA-damaging process.