2D materials possess many interesting properties, and have shown great application potentials. In this work, the development of humidity-responsive, 2D plasmonic nanostructures with switchable chromogenic properties upon wetting–dewetting transitions is reported. By exploiting DNA hybridization-directed anchoring of gold nanoparticles (AuNPs) on substrates, a series of single-nanoparticle-layer (SNL) plasmonic films is fabricated. Due to the collective plasmonic responses in SNL, these ultrathin 2D films display rapid and reversible red-blue color change upon the wetting–dewetting transition, suggesting that hydration-induced microscopic plasmonic coupling between AuNPs is replicated in the macroscopic, centimeter-scale films. It is also found that hydration finely tunes the electric field distribution between AuNPs in the SNL film, based on which responsive surface-enhanced Raman scattering substrates with spatially homogeneous hot spots are developed. Thus it is expected that DNA-mediated 2D SNL structures open new avenues for designing miniaturized plasmonic nanodevices with various applications.