G-Quadruplexes (G4s) are thermodynamically stable, compact, and poorly hydrated structures that pose a potent obstacle for chromosome replication and gene expression, and requiring resolution by helicases in a cell. Bulk stopped-flow fluorescence assays have provided many mechanistic insights into helicase-mediated duplex DNA unwinding. However, to date, detailed studies on intramolecular G-quadruplexes similar or comparable with those used for studying duplex DNA are still lacking. Here, we describe a method for the direct and quantitative measurement of helicase-mediated intramolecular G-quadruplex unfolding in real time. We designed a series of site-specific fluorescently double-labeled intramolecular G4s and screened appropriate substrates to characterize the helicase-mediated G4 unfolding. With the developed method, we determined, for the first time to our best knowledge, the unfolding and refolding constant of G4 (≈ 5 s-1), and other relative parameters under single-turnover experimental conditions in the presence of G4 traps. Our approach not only provides a new paradigm for characterizing helicase-mediated intramolecular G4 unfolding using stopped-flow assays but also offers a way to screen for inhibitors of G4 unfolding helicases as therapeutic drug targets. Graphical abstract.