Pentatricopeptide repeat proteins (PPR) are a large family of modular RNA-binding proteins that have a plethora of roles in transcription regulation, RNA-processing, splicing, stability and translation. PPRs are relatively unique amongst RNA-binding proteins in that they bind to specific ssRNA target sequences; their specificity is ultimately determined by repeating modular motifs, whereby each consecutive module is responsible for the specific recognition of a discrete RNA nucleotide. As such, there is significant potential to develop ‘designer’ PPRs for use in diagnostics and as a tool to detect and localize target RNA sequences in vivo. However, questions remain regarding the binding kinetics of PPR to its target RNA and whether the presence of RNA mismatches or secondary structure affect the affinity or specificity of PPR to RNA. With this in mind, we developed a PPR biosensor that can report on ssRNA binding via a measurable change in fluorescence resonance energy transfer (FRET). Using this biosensor, we perform single-molecule FRET (smFRET) experiments that allow the binding of ssRNA constructs to a single PPR protein to be monitored in real time and the kinetics quantified. We observe that PPR binds to its target sequence with high affinity (~ 10 nM), however, the introduction of even a single mismatch to the target ssRNA sequence can significantly decrease PPR association rates and increase dissociation rates. Furthermore, longer ssRNA sequences were observed to have significantly slower association rates. Finally, to confirm that the changes in FRET that we observe are due to conformational rearrangement of PPR upon ssRNA binding, we performed 3-colour smFRET experiments with the PPR biosensor and a fluorescently-labelled ssRNA oligonucleotide. Indeed, association of the labelled-ssRNA was observed to directly correlate with PPR conformational changes. In summary, we have developed a system that enables the molecular mechanisms of PPR-ssRNA interactions to be determined.