Biological species, including reactive oxygen species (ROS), reactive sulfur species (RSS), reactive nitrogen species (RNS), F−, Pd2+, Cu2+, Hg2+, and others, are crucial for the healthy functioning of cells in living organisms. However, their aberrant concentration can result in various serious diseases. Therefore, it is essential to monitor biological species in cellular organelles such as the cell membrane, mitochondria, lysosome, endoplasmic reticulum, Golgi apparatus, and nucleus. Among various fluorescent probes for species detection within the organelles, ratiometric fluorescent probes have drawn special attention as a potential way to get beyond the drawbacks of intensity-based probes. This method depends on measuring the intensity change of two emission bands (caused by an analyte), which produces an efficient internal referencing that increases the detection's sensitivity. This review article discusses the literature publications (from 2015 to 2022) on organelle-targeting ratiometric fluorescent probes, the general strategies, the detecting mechanisms, the broad scope, and the challenges currently faced by fluorescent probes.