In vitro transcription and translation reactions have become popular for a bottom-up approach to synthetic biology. Concentrations of the mRNA intermediate are rarely determined, although knowledge of synthesis and degradation rates could facilitate rational engineering of in vitro systems. We designed binary probes to measure mRNA dynamics during cell-free protein synthesis by fluorescence resonance energy transfer. We tested different mRNA variants and show that the location and sequence environment of the probe target sites are important parameters for probe association kinetics and output signal. Best suited for sensitive real-time quantitation of mRNA was a target site located in the 3' untranslated region, which we designed to reduce secondary structure. We used this probe-target pair to refine our knowledge of mRNA dynamics in the commercially available PURE cell-free protein synthesis system and characterized the effect of TetR repressor on mRNA synthesis rates from a T7 promoter.