Photoswitch-modified DNA is being studied for applications including light-harvesting molecular motors, photocontrolled drug delivery, gene regulation, and optically mediated assembly of plasmonic metal nanoparticles in DNA-hybridization assays. We study the sequence and hybridization dependence of the photoisomerization quantum yield of azobenzene attached to DNA via the popular d-threoninol linkage. Compared to free azobenzene we find that the quantum yield for photoisomerization from trans to cis form is decreased 3-fold (from 0.094 ± 0.004 to 0.036 ± 0.002) when the azobenzene is incorporated into ssDNA, and is further reduced 15-fold (to 0.0056 ± 0.0008) for azobenzene incorporated into dsDNA. In addition, we find that the quantum yield is sensitive to the local sequence including both specific mismatches and the overall sequence-dependent melting temperature (Tm). These results serve as design rules for efficient photoswitchable DNA sequences tailored for sensing, drug delivery, and energy-harvesting applications, while also providing a foundation for understanding phenomena such as photonically controlled hybridization stringency.