The maintenance and regulation of protein homeostasis is heavily dependent on a vast network of molecular chaperones, which together act to prevent the formation and accumulation of misfolded and aggregated forms of proteins. Interactions between chaperone proteins and their clients are often transient and heterogeneous in nature, which make them difficult to study using traditional bulk-phase measurements due to ensemble averaging. Recently, single-molecule techniques have emerged as a powerful tool to study chaperone function since individual proteins can be monitored in real time, enabling the characterization of rare and transient species that may be present. Consequently, we aimed to develop a protein folding-sensor based on the rhodanese and firefly luciferase proteins that can be used in single-molecule experiments to report on folding transitions upon interaction with chaperones. To do so, the soluble expression and purification of rhodanese and luciferase cysteine variants was optimised and the purified protein subsequently labelled with fluorophore pairs that enable the folded state to be monitored via single-molecule fluorescence resonance energy transfer (smFRET). smFRET analysis revealed that individual rhodanese and luciferase molecules exhibited high FRET efficiencies when correctly folded (~ 0.8 E and ~ 0.7 E, respectively) and exhibit much lower FRET efficiencies (~ 0.2 E) when chemically unfolded. Crucially, through the use of microfluidics, transitions between folded (high FRET) and unfolded (low FRET) conformations can be monitored on the millisecond timescale in response to changing denaturant conditions. Collectively, these results suggest that both rhodanese and luciferase could be implemented as folding-sensors in single-molecule experiments to study chaperone function.