We report a label-free optical detection scheme for DNA hybridization using photon upconverting nanoparticles. On the basis of luminescence resonance energy transfer between a donor and an acceptor, the method is highly sensitive and can differentiate targets with single base variation. Photon upconverting nanoparticles were used as the donor and an intercalating dye as the acceptor. The sensor could differentiate the perfectly matched target from the single-base mismatched target. The detection limit of this sensor toward perfectly matched target was calculated to be 20 fmol, with no photobleaching. Oligonucleotide sensors of such design demonstrate high sensitivity and specificity without fluorophore labeling.