Intrinsic molecular brightness (MB) is a number of emitted photons per second per molecule. When a substrate labeled by a fluorophore and a second unlabeled substrate form a complex in solution, the MB of the fluorophore changes. Here we use this change to determine the equilibrium constant (K) for the formation of the complex at pM concentrations. To illustrate this method, we used a reaction of DNA hybridization, where only one of the strands was fluorescently labeled. We determined K at the substrate concentrations from 80 pM to 30 nM. We validated this method against Förster resonance energy transfer (FRET). This method is much simpler than FRET as it requires only one fluorophore in the complex with a very small (a f̃ew percent) change in MB.