Abstract: This dissertation presents a major advance in single-molecule methodology and its application in investigating protein folding and polymer physics. Single-molecule Forster Resonance Energy Transfer (FRET) technique was combined with time-correlated single photon counting to implement nano-second Alternating Laser EXcitation (ns-ALEX), a novel fluorescence technique in biophysics and life sciences. The instrumentation construction, photon acquisition algorithm and data analysis procedures were described in full details. This technique was then used to extract rich information in the polymer behavior of macro molecules. By modeling distance distribution in corresponding systems including denatured proteins and polyelectrolytes, ns-ALEX was demonstrated to yield otherwise inaccessible information on the conformational variations of biomacromolecules. Furthermore, ns-ALEX was coupled with direct-modulation ALEX, and applied in deciphering the photophysical pathways of generally used fluorophores.