Drug discovery is a complex process in which many challenges need to be overcome, from the discovery of a drug candidate to ensuring the efficacy and safety of the candidate in humans. Modern analytical methods allow tens of thousands of drug candidates to be screened for their inhibition of specific enzymes or receptors. In recent years, fluorescent probes have been used for the detection and diagnosis of human pathogens as well as high-throughput screening. This review focuses on recent progress in organic small-molecule based enzyme-activated fluorescent probes for screening of inhibitors from natural products. The contents include the construction of fluorescent probes, working mechanism and the process of inhibitor screening. The progress suggests that fluorescent probes are a vital and rapidly growing technology for inhibitor screening of enzymes, in particular, inhibitor screening in situ.