Despite recent progress in 2D nanomaterials-based biosensing, it remains challenging to achieve sensitive and high selective detection. This study develops few-layer graphdiyne (GD) nanosheets (NSs) that are used as novel sensing platforms for a variety of fluorophores real-time detection of DNA with low background and high signal-to-noise ratio, which show a distinguished fluorescence quenching ability and different affinities toward single-stranded DNA and double-stranded DNA. Importantly, for the first time, a few-layer GD NSs-based multiplexed DNA sensor is developed.