The process of unwinding and renaturation of DNA has been widely used in studies of nucleotide sequence organization. Compared with traditional methods for DNA unwinding and renaturation, the label-free and non-destruction detection technology is significant and desiderated. We realized an optical system based on optical rotation via weak measurement for detection of single- and double-strand state of DNA. The optical rotation, which was induced by the status change of single and double DNA strands, was exploited to modulate the preselected polarization of a weak measurement system. With this modulation, the optical rotation caused by the separation of DNA strands can be determined through the center wavelength shift of the output spectrum. By monitoring the wavelength shift in real time, the separation processes of the DNAs with different base ratio (25% and 70%) and length (4nt and 40nt), and DNAs with three terminally modified cholesterol molecules were experimentally explored in varied pH and temperature conditions. In addition, the detection limit of the DNA concentration was obtained to be 5 × 10-6 mol/L. Our work based on optical rotation detection of single- and double-strand DNA exhibits the unique advantages of real-time monitoring, label-free, non-destruction and simplicity.