Magnetic resonance spectroscopy of single biomolecules under near-physiological conditions may substantially advance understanding of biological function, yet remains very challenging. Here we use nitrogen-vacancy centers in diamonds to detect electron spin resonance spectra of individual, tethered DNA duplexes labeled with a nitroxide spin label in aqueous buffer solutions at ambient temperatures. This paves the way for magnetic resonance studies on single biomolecules and their inter-molecular interactions in a native-like environment.