Recent advances in single-molecule detection and single-molecule spectroscopy at room temperature by laser-induced fluorescence offer new tools for the study of individual macromolecules under physiological conditions. These tools relay conformational states, conformational dynamics, and activity of single biological molecules to physical observables, unmasked by ensemble averaging. Distributions and time trajectories of these observables can therefore be measured during a reaction without the impossible need to synchronize all the molecules in the ensemble. The progress in applying these tools to biological studies with the use of fluorophores that are site-specifically attached to macromolecules is reviewed.