Ultrasensitive and in situ DNA detection at different pH values, ranging from 4.3 to 8.5, based on a microfiber with a graphene oxide linking layer was proposed and experimentally demonstrated. The graphene oxide coating over the silica microfiber provides a strong π–π interaction with the detected single-stranded DNA molecules, and induces an amplified surface refractive index modulation over the fiber, and finally leads to a wavelength shift of the optical interference fringe for interrogation. Using this strategy, in situ DNA measurement with a detection limit of up to 10⁻¹² M and a linear response in the pH range from 4.3–8.5 has been achieved. Benefiting from its compact size, high sensitivity, and ease of use, together with remote operational ability, the proposed sensor opens up a multitude of opportunities for quantifying DNA in various hard-to-reach environments. It may supplement the existing DNA detection tools.