Layer-by-layer self-assembled multilayer thin films of gold nanoparticles (GNPs) linked with myoglobin (Mb) show substantial sensitivity to humidity at room temperature according to measurements of localized surface plasmon resonance (LSPR) absorption that relies on the interparticle interaction present in the film. The sensor response is reversible, with response and recovery times as low as 5 s. The sensing mechanism is as follows: as the ambient humidity changes, Mb molecules change their size, making the GNP-to-GNP spacing and thereby the interparticle interaction change; the change in the interparticle interaction causes a change in the LSPR absorption of the multilayer thin film. We found that the LSPR band of the multilayer thin film was almost insensitive to both the surrounding refractive index and the adlayer thickness, rendering the multilayer-film-based humidity sensor highly immune to ambient disturbances.