Abstract Microenvironment-sensitive fluorescent nucleosides are highly important for biotechnological applications. Here, we report the synthesis and photophysical properties of various new 8-phenylethynylated 2′-deoxyadenosines and 2′-deoxyisoguanosines. 8-phenylethynylated 2′-deoxyadenosines are synthesized using Sonogashira cross-coupling reaction (76–91%), which are further transformed to 8-phenylethynylated 2′-deoxyisoguanosines (50–63%). All new compounds exhibit strong solvatochromicity and pH-dependent fluorescence in aqueous solutions. Furthermore, 8-phenylethynylated purine analogues are sensitive to solvent viscosity changes, indicating their potential sensitivity to environmental changes.