Fluorescent probes targeting proteins are used to investigate biological processes, requiring strong binding affinity and favorable fluorescence. In this study, we present the first fluostere with optimized fluorescence properties. We started exploring the fluorescence of acidic pyrazolo[1,5-a]pyridin-2-ol, and, by the introduction of EWGs, π-conjugation, incorporation of push-pull systems and rigid structures, we optimized emission profiles and QY, providing a first Structure-Fluorescence relationship (SFR) of the system. To provide proof of concept in biological applications, the established SFR was integrated with hDHODHi, an important oncology target, enabling the SAR designing fluorescent hDHODHi 11a and 14, with 11a being the most potent IC50 = 170 nM. These inhibitors were validated in vitro for their antileukemic and antiviral activity. As they are both environmentally sensitive fluorescent probes that can highlight their binding to the target, their fluorescence was found to colocalize in the mitochondria, where hDHODH is located, in cellular experiments.