The maintenance and regulation of protein homeostasis is heavily reliant on the Hsp40/Hsp70 system of molecular chaperones, which play critical roles in de novo protein folding and the refolding of misfolded proteins. However, due to the dynamic and heterogeneous nature by which these chaperones interact with their co-chaperones and clients, questions remain regarding the precise molecular mechanisms by which Hsp70 affects the conformation of their clients and assists in their refolding. To address this, we have developed the chaperone client protein, firefly luciferase (Fluc), such that its conformation can be monitored temporally using single-molecule fluorescence resonance energy transfer (smFRET) and total internal reflection fluorescence (TIRF) microscopy. For the first time, the conformation of individual client proteins as they are being folded by the bacterial or human Hsp70 chaperone machinery (i.e., Hsp40, Hsp70 and a nucleotide-exchange factor) was monitored in real time. smFRET results revealed that Hsp40 binds to and partially unfolds the client for delivery to Hsp70. Binding of the client by Hsp70 results in additional client expansion, thereby resolving misfolded states, which provides an opportunity for correct folding upon chaperone release. Moreover, we demonstrate that multiple cycles of chaperone binding-and-release to a single misfolded client protein is conducive for efficient refolding. Crucially, the temporal observation of the chaperone-assisted refolding process enabled key kinetic details to be determined that are typically inaccessible using other approaches. Thus, the developed Fluc protein-folding sensor is an ideal tool and represents an exciting platform for further single-molecule investigations of chaperone function.