Herein, we report our work exploring the essential requirements for fluorophore selection during the development of various fluorescence applications. We assembled a library of chromone-derived fluorophores with diverse structure-fluorescence properties, which allowed us to choose the fluorophore pairs with similar structures but differing fluorescence properties and compared the performance of the selected fluorophore pairs in three types of commonly used fluorescence applications. We found that the selection standard of a suitable fluorophore is variable depending on the application. (1) In fluorescence imaging, fluorophores with strong and constant fluorescence under various conditions, such as a large pH range, are preferred. Notably, (2) in the detection of bioactive species, fluorophores with relatively lower fluorescence quantum yield favor the detection sensitivity. Furthermore, (3) in enzymatic assays employing fluorescence, the key parameter is the binding affinity between the fluorophore and the enzyme.