In chemical biology the concept of chemosensors and chemical probes implicates the transduction of a chemical signal upon binding with the chemical substance. There are enormous challenges for developing superior chemical probes using the target-oriented synthesis approach with limited knowledge of the recognition site. The emerging field of the diversity-oriented fluorescent library approach (DOFLA) enables a conceptually novel and new strategy for designing chemosensors/probes. Vast chemical diversity in optically active fluorophores provides limitless opportunity for probe development, likewise drug discovery from diversity-oriented synthesis approaches. Synergistic effects of diverse chemical structures, high-throughput screening methodology, and improved microscopic automation have led to several chemical probes in the last two decades. This chapter summarizes the recent developments of chemical probes/sensors and chemosensors using DOFLA and their biological and environmental applications. The chapter consists of six sections, including the concept of the diversity-oriented fluorescent library, mechanisms of signal transduction, screening strategies, and reviews of recent examples from in vitro spectra-based screening, cell-based screening, and in vivo whole organelle-based screening.