Enzymes are a class of important substances for life, and their abnormal levels are associated with many diseases. Thus, great progress has been made in the past decade in detecting and imaging enzymes in living biosystems, and in this respect fluorescent probes combined with confocal microscopy have attracted much attention because of their high sensitivity and unrivaled spatiotemporal resolution. Fluorescent probes are usually composed of three moieties: a signal or fluorophore moiety, a recognition or labeling moiety, and an appropriate linker to connect the two aforementioned moieties. At present, however, research and reviews on enzymatic probes mostly focus on fluorophores and/or linkers, whereas those on the recognition moiety are relatively few. Moreover, current enzymatic probes with some recognition moieties have drawbacks such as poor selectivity, high background fluorescence, or/and low sensitivity and are unsatisfactory for practical applications. Thus, developing new recognition moieties with higher specificity or/and sensitivity to the enzyme of interest is very desirable but still challenging. In this Account, we introduce the recognition moieties of fluorescent probes for several enzymes, including tyrosinase, monoamine oxidase A (MAO-A), nitroreductase (NTR), and aminopeptidases. Highlights are given on how new specific recognition moieties of tyrosinase and MAO-A were designed to eliminate the interference by reactive oxygen species (ROS) and MAO-B, respectively. Here we present four recent examples in which designed fluorescent probes are employed to image enzymes in living biosystems. The first example shows that 3-hydroxyphenyl can serve as a new and more specific recognition moiety than the traditional 4-hydroxyphenyl group for tyrosinase, enabling the development of a highly selective fluorescent probe for imaging of tyrosinase without interference by ROS. The second presents a general design strategy for fluorescent probes specific for an enzyme, which involves combining the characteristic structure of an inhibitor of the target enzyme along with its traditional reactive group as a new recognition moiety, and successfully demonstrates it by selective detection of MAO-A in the presence of its isomeric MAO-B. The third mainly illustrates that 5-nitrothiophen-2-yl alcohol with a stronger electron-donating S atom is a better fluorescence quenching and recognition moiety than 5-nitrofuran-2-yl alcohol for NTR, leading to the development of a highly sensitive method for NTR assay. Lastly, on the basis of known observations, we show that besides the specific interaction with the target, another function of some recognition moieties may be responsible for tuning the fluorescence signal, which is exemplified by the linking of several aminopeptidases' recognition moieties to the free hydroxyl or amino group of different fluorophores. It is our wish that this Account will promote the appearance of more specific recognition moieties and fluorescent probes with excellent properties and that new biofunctions of the enzymes will be uncovered.