Synthetic fluorescent probes have emerged as versatile tools that can help researchers to gain insight into the roles of biomolecules in various diseases. Designing and discovering selective chemical probes for biomolecules has become a topic of active investigation in recent years. In this talk, I will present several examples to illustrate how synthetic fluorescent probes are designed and utilized for biological investigation. HDACs play important roles in regulating various cellular functions. We have designed and synthesized a number of fluorescent probes for selective detection of the enzymatic activity of HDACs. The designed probes are capable of detecting HDAC activity in a continuous fashion, eliminating the extra step of protease cleavage. Further development of dual-function probe will not only enable reporting of enzymatic activity, but also allow us to identify and capture protein targets from the complex cellular environment. We have extended this strategy to detect enzymatic activity of decrotonylase, desuccinylase and demethylase, etc. In another study, we systematically investigated the rhodamine intramolecular spirocyclization switching mechanism and discovered new rhodamine fluorophores for super-resolution imaging. The fluorophores were successfully utilized for investigating mitochondria morphology change with nanoscale resolution. We envision that the continued evolution of synthetic probes holds promise for investigating the intricate cellular processes and advancing disease diagnosis and therapeutic development.