RNA and DNA play distinct roles in biological systems. However, the underlying physicochemical difference has been poorly understood, in particular, that in dynamical aspects. In this paper, we report on a comparative study of the formation−dissociation dynamics of a hairpin structure of RNA and DNA with development of two-color two-dimensional fluorescence lifetime correlation spectroscopy (two-color 2D FLCS). In this extension of 2D FLCS, we newly introduce the two-color detection scheme to analyze not only donor fluorescence photons but also acceptor fluorescence photons from a doubly labeled Forster resonance energy transfer (FRET) pair. This new 2D FLCS is utilized to resolve multiple species present in an equilibrated condition with a microsecond time resolution and enhanced sensitivity, and the combined use with the filtered fluorescence correlation spectroscopy (FCS) method enables a quantitative discussion on microsecond structural dynamics occurring in the equilibrium. This integrated approach is applied to FRET-labeled RNA/DNA oligonucleotides having analogous hairpin-forming sequences, and it was revealed that the hairpin dissociation rate of RNA is an order of magnitude slower than that of DNA while their hairpin-forming rates are comparable. This marked difference is attributable to the distinct duplex structure of RNA and DNA. The present study demonstrates that the integrated approach combining two-color 2D FLCS and filtered FCS has a high potential for quantifying microsecond kinetics at the single-molecule level, which allows us to experimentally construct a free energy landscape.