The specific interaction of the RNA recognition motif of Rev and its viral mRNA target, RRE, has been demonstrated for the first time at the single-molecule level by atomic-force-microscope based single-molecule-force-spectroscopy (AFM-SMFS). The approach reveals details of the dissociation pathway and contribution of base mutations. Specific RNA–protein interaction is efficiently blocked by the RNA binding agent neomycin, showing the potential of AFM-SMFS as an efficient tool for single-molecule drug screening of RNA targets. Furthermore, we show the importance of surface chemistry in AFM-SMFS of RNA–protein interaction, in particular the influence of polymer linkers.