Organelles perform critical biological functions due to their distinct molecular composition and internal environment. Disorders in organelles or their interacting networks have been linked to the incidence of numerous diseases, and the research of pharmacological actions at the organelle level has sparked pharmacists' interest. Currently, cell imaging has evolved into a critical tool for drug delivery, drug discovery, and pharmacological research. The introduction of advanced imaging techniques in recent years has provided researchers with richer biological information for viewing and studying the ultrastructure of organelles, protein interactions, and gene transcription activities, leading to the design and delivery of precision-targeted drugs. Therefore, this reviews the research on organelles-targeted drugs based upon imaging technologies and development of fluorescent molecules for medicinal purposes. We also give a thorough analysis of a number of subcellular-level elements of drug development, including subcellular research instruments and methods, organelle biological event investigation, subcellular target and drug identification, and design of subcellular delivery systems. This review will make it possible to promote drug research from the individual/cellular level to the subcellular level, as well as give a new focus based on newly found organelle activities.