Fluorogenic RNAs that are based on the complex formed by 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) derivatives and the RNA aptamer named Spinach were used to engineer a new generation of in vitro and in vivo sensors for bioanalytics. With the resolved crystal structure of the RNA/small molecule complex, the engineering map becomes available, but comprehensive information regarding the thermodynamic profile of the molecule is missing. Here, we reconstructed the full thermodynamic binding and stability landscapes between DFHBI and a truncated sequence of first-generation Spinach. For this purpose, we established a systematic screening procedure for single- and double-point mutations on a microfluidic large-scale integrated chip platform for 87-nt long RNAs. The thermodynamic profile with single base resolution was used to engineer an improved fluorogenic spinach generation via a directed rather than evolutional approach. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.