Rapid, sensitive, and reliable nucleic acid assay is crucial for the molecular diagnosis of many diseases. For high sensitivity, conventional techniques require time-consuming, high-cost, and complicated procedures, such as enzymatic gene amplification, labeling, and purification, limiting their applications to point-of-care diagnostics. Herein we report a new DNA nanoprobe based on the dual effects of target-specific plasmon-enhanced fluorescence and off-target plasmonic quenching. Janus gold half-shell/polystyrene nanospheres (hsAu/PSs, similar to 150 nm diameter) are tethered with capture single-stranded DNA (ssDNA), coupled with a fluorophore-conjugated reporter ssDNA through sandwich-type hybridization with target DNA, resulting in 5-fold increase through plasmon-enhanced fluorescence. Smaller gold nanoparticles (similar to 13 nm diameter) are subsequently introduced as quenchers to reduce background fluorescence from unhybridized reporter ssDNA, increasing the sensitivity about 10(3) times. The limit of detection of the dual-mode plasmonic DNA nanoprobe is 16 pM at room temperature in 1 h for the target gene of Klebsiella pneumoniae carbapenemase. The nanoprobe also exhibits a high selectivity enough to discriminate a single-base difference in the target gene. Our strategy harnesses both of the plasmonmediated fluorescence enhancement and quenching effects through the sophisticated design of nanoscale colloids, which opens a promising avenue to the enzyme-free, simple, sensitive, and selective detection of pathogenic DNA.