Helicases are ubiquitous ‘molecular motor’ enzymes that disrupt nucleic acid (NA) helices and NA-protein interactions. Despite the key roles of helicases in many cellular processes and diseases, their target repertoires and the determinants of their functional specialization are often uncertain. We have developed a new enzymatic screening scheme based on NA duplex unwinding, Helicase-SELEX, to find natural or synthetic helicase substrates in large NA sequence libraries and probe specificity and cofactor requirements at global scale. Using the transcription termination Rho helicase as a prototype for Helicase-SELEX, we have discovered ~3300 functional substrate sequences in Escherichia coli, thereby providing the first detailed map of Rho utilization (Rut) sites at genome scale. Moreover, we have determined that inclusion of a cofactor (NusG) in the Helicase-SELEX scheme can modulate the selection outcome and help identify specificity determinants. Hence, the Helicase-SELEX maps reveal many new features, including promoter- and intrinsic terminator-associated Rut sites, bidirectional Rut tandems, and NusG-dependent Rut sites with unusual G>C skewed compositions. In a parallel effort, we successfully implemented a variant of Helicase-SELEX to evolve synthetic Rut sequences able to elicit Rho activity in vitro and in vivo only in presence of an orthogonal cofactor (serotonin). Thus, H-SELEX is a unique tool to evolve synthetic riboswitches from scratch and grant access to a new type of synthetic regulation based on helicase function. Altogether, our data illustrate the power and flexibility of Helicase-SELEX to seek constitutive or conditional helicase substrates in natural or synthetic NA libraries for fundamental or synthetic biology discovery.