Polyelectrolyte brushes (PEBs) undergo conformational transitions due to changes in pH and/or ionic strength, which is leveraged as smart surfaces and on-demand drug-release systems. However, probing conformational transitions of functional PEBs has remained challenging due to low spatiotemporal resolution of characterization methods. Herein, fluorescently-coupled PEBs are devised that give rise to Förster Resonance Energy Transfer (FRET) intrinsically coupled to conformational transitions of chains. Polyelectrolyte poly(2-(dimethylamino)ethyl methacrylate) brushes are grown on silica surfaces via a grafting-from approach, producing nanoscale brushes ≈60 nm in solvated height. The study chose to investigate pH as astimulus, at constant ionic strength, using pH-insensitive fluorophores coupled within the brush (donors) and on-chain ends (acceptors), leading to conformational FRET. The influence of pH on the FRET brushes is proved by ellipsometry and fluorescence spectroscopy. Importantly, using FRET meant chain conformation is spatially resolved with sub-micrometer resolution by confocal laser scanning microscopy, where subtle changes in brush conformation are resolved in seconds. Unique mixing dynamics of different pH microdroplets on the brushes are identified as coalescence occurred, with reversible output, and a clear delay in brush responses to mixing liquids. The surfaces offer a new basis for probing conformational transitions of PEBs with high spatiotemporal resolution.