Water-soluble nano-C60 can serve as a novel, effective, fluorescent sensing platform for biomolecular detection with high sensitivity and selectivity. In this paper, fluorescent detection of DNA and thrombin via nano-C60 is demonstrated for the first time. The principle of the assay lies in the fact that the adsorption of the fluorescently labeled single-stranded DNA (ssDNA) probe by nano-C60 leads to substantial fluorescence quenching. In the presence of a target, the biomolecular mutual interaction suppresses this quenching, signaling the existence of the target. This sensing system rivals graphene oxide but is superior to other carbon-structure-based systems. The present method can also achieve multiplex DNA detection and withstand the interference from human blood serum.