Abstract Pentamethyl cyanine dyes are promising fluorophores for fluorescence sensing and imaging. However, advanced biomedical applications require enhanced control of their excited‐state properties. Herein, a synthetic approach for attaching aryl substituents at the C2’ position of the thio‐pentamethine cyanine (TCy5) dye structure is reported for the first time. C2’‐aryl substitution enables the regulation of both the twisted intramolecular charge transfer (TICT) and photoinduced electron transfer (PET) mechanisms to be regulated in the excited state. Modulation of these mechanisms allows the design of a nitroreductase‐activatable TCy5 fluorophore for hypoxic tumor photodynamic therapy and fluorescence imaging. These C2’‐aryl TCy5 dyes provide a tunable platform for engineering cyanine dyes tailored to sophisticated biological applications, such as photodynamic therapy.