Stimuli-responsive polymeric nanoparticles have the potential to radically enhance the delivery of therapeutic cargo including peptides, proteins, RNA, DNA, and other biomacromolecules. To improve the efficiency of these drug delivery systems, accurate knowledge of their structural dynamics, disassembly process, and loading/release behavior is vital. Amongst a myriad of fluorescent probes utilized for this purpose, environmentally responsive fluorophores demonstrate distinctive advantages due to significant changes in fluorescence intensity, lifetime and/or emission wavelength with variation in their environment. In this work, we designed a series of novel multifunctional probe molecules, isoquinoline betaines (IQBs), with exquisite solvatofluorochromic properties. Through both a steady absorption signal in the visible wavelength range, and an environmentally dependent emission, these IQBs are a powerful tool for simultaneously tracking multiple key processes, including nanoparticle formation and disassembly, the loading and distribution of drug molecules, and the responsive release of drugs. This novel fluorescent probe was covalently conjugated to a pH-responsive nanoparticle and successfully probed the nanoparticle’s internal structural rearrangement while also monitoring its drug-release activity of a model peptide in real-time. This IQB fluorescent probe system enhances our understanding of how nanoparticles interact with both their cargo and microenvironment and thus represents an important step forward in the development of more efficient drug delivery systems.