The formation and maintenance of single-stranded DNA (ssDNA) are essential parts of many processes involving DNA. For example, strand separation of double-stranded DNA (dsDNA) is catalyzed by helicases, and this exposure of the bases on the DNA allows further processing, such as replication, recombination, or repair. Assays of helicase activity and probes for their mechanism are essential for understanding related biological processes. Here we describe the development and use of a fluorescent probe to measure ssDNA formation specifically and in real time, with high sensitivity and time resolution. The reagentless biosensor is based on the ssDNA binding protein (SSB) from Escherichia coli, labeled at a specific site with a coumarin fluorophore. Its use in the study of DNA manipulations involving ssDNA intermediates is demonstrated in assays for DNA unwinding, catalyzed by DNA helicases.