Although the hydrogen bond is one of the weakest chemical interactions, it plays a key role in forming Watson–Crick base pairs (adenine–thymine and guanine–cytosine) that hold together the two helical chains of nucleotides in DNA molecules and form the basis of the genetic code. [1–3] The bonding process is easily influenced by molecular structures, steric hindrance, [4] the base-pair geometry, [5] and the surrounding environment. [5–7] X-ray crystal structure analysis and NMR spectroscopy, [8] infrared spectroscopy, [9–11] Raman spectroscopy, [12] electrochemistry [13] and the quartz crystal microbalance technique [14] have been employed to explain the basic biological, chemical and physical mechanisms of hydrogen bonding. Most of the methods are based on the data collected from large amounts of molecules. Due to its extremely weak chemical interaction, hydrogen bonding gives rather subtle signals that are easily covered by signals from other stronger interactions between the molecules. Highly sensitive and selective methods are needed to get more accurate insight into hydrogen bonding. Tip-enhanced Raman spectroscopy (TERS) is a recently developed near-field spectroscopic method, which can simultaneously achieve high spatial resolution from the scanning probe microscopy (SPM) images and rich chemical information from the Raman spectra. [15, 16] Benefitting from the greatly enhanced electromagnefic field generated in the cavity of the SPM tip and the substrate by illuminating the SPM tip, TERS has been successfully applied to Raman studies on atomically smooth surfaces with high reproducibility. [16] The sample volume in surface-enhanced Raman spectroscopy (SERS) is defined by the laser focal spot projected on a rough coinage metal surface, that is, about 1 to 2 mm 2 ( 10 12 m 2 ) from where large amounts of molecules are excited. By using an STM tip to focus the enhanced electromagnetic field into an area of ~ nm range (~ 10 15 m 2 for a tip of 40 nm in diameter) [17] at an atomically smooth surface, the number of the excited molecules is dramatically reduced, allowing one to study molecular events at approximately the single-molecule level for resonant molecules [17] and picomole level for non-resonant molecules, such as DNA bases. [18] Very recently, a single brilliant cresyl blue molecule has been spectroscopically detected and imaged by using the UHV–TERS setup in our group. [19] A sensitive study of hydrogen bonding can therefore be achieved at single crystalline surfaces without information-averaging between large amounts of molecules and interference from ill-defined substrates.