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Recently developed single-molecule spectroscopy (SMS) permits the analysis of fluorescent mixtures one molecule at a time. SMS methods provide the means to make rapid measurements on small, complex samples without the need for separations and target amplification enabling a new class of ultrasensitive nucleic acid assays. Here we give a brief overview of the current state of the art of SMS nucleic acid analysis and discuss ongoing work in our laboratory on two-color single-molecule fluorescence detection of specific nucleic acid sequences. In the future, two-color SMS nucleic acid assays will be used for a variety of applications including: gene expression analysis, disease detection and genomics.