Small molecular fluorescent probe technology has become a potential tool for biosensing and bioimaging since it can realize real-time dynamic tracking and monitoring of active molecules and microenvironment changes in living organisms with advantages of less disturbance to biological samples, extremely high sensitivity and specificity. In this review, we summarize some characteristics of cellular microenvironment and related bioactive molecules commonly found in them. The design strategies of the molecular fluorescent probes utilized to monitor changes of cellular microenvironments and active molecules are also discussed. Some recently developed molecular fluorescent probes used to monitor microenvironmental changes and active molecules in organisms have also been listed in this review. Additionally, sensing behavior and potential application of these fluorescent probes have also been discussed.