Monitoring the site-specific local structure and dynamics of polynucleotides and DNA is important for understanding their biological functions. However, structurally characterizing these biomolecules with high time resolution has been known to be experimentally challenging. In this work, several 5-silylethynyl-2′-deoxynucleosides and 5-substituted phenylethynyl-2′-deoxynucleosides on the basis of deoxycytidine (dC) and deoxythymidine (dT) were synthesized, in which the alkynyl group shows intensified CC stretching vibration with infrared transition dipole moment magnitude close to that of typical CO stretching, and exhibits structural sensitivities in both vibrational frequency and spectral width. In particular, 5-trimethylsilylethynyl-2′-dC (^TMSEdC, molecule 1a) was examined in detail using femtosecond nonlinear IR spectroscopy. The solvent dependent CC stretching frequency of 1a can be reasonably interpreted mainly as the hydrogen-bonding effect between the solvent and cytosine base ring structure. Transient 2D IR and pump–probe IR measurements of 1a carried out comparatively in two aprotic solvents (DMSO and THF) and one protic solvent (MeOH) further reveal solvent dependent ultrafast vibrational properties, including diagonal anharmonicity, spectral diffusion, vibrational relaxation and anisotropy dynamics. These observed sensitivities are rooted in an extended π-conjugation of the base ring structure in which the CC group is actively involved. Our results show that the intensified CC stretching vibration can potentially provide a site-specific IR probe for monitoring the equilibrium and ultrafast structural dynamics of polynucleotides.