The ability to tune the sensing properties with nanostructured materials in a flexible scaffold is essential for constructing highly sensitive and wearable sensors or biosensors. Here we demonstrate a novel class of nanoparticle-nanofibrous membranes as a tunable interfacial scaffolds for flexible sweat sensors by assembling gold nanoparticles (Au NPs) in a multilayered fibrous membrane consisting of cellulose nanofiber (CN) top layer (fiber diameter 5 nm), electrospun polyacrylonitrile (PAN) nanofibrous midlayer (fiber diameter 150 nm), and nonwoven polyethyleneterephthalate (PET) fibrous support layer (fiber diameter 20 pm) through interparticle molecular/polymeric linkages and nanoparticle-nanofibrous interactions. One strategy involves 11-mercaptoundecanoic acid (MUA) as a molecular linker having hydrogen-bonding groups for interlinking alkanethiolate-capped Au NPs, and the other features poly(diallyldimethylammonium) (PDA) as a matrix with positively changed groups for anchoring negative-charge capped Au NPs. Impedance measurements of the nanocomposite membrane (Au NPs/CN/PAN/PET) as a scaffold on chemiresistor-type platforms have demonstrated the viability of detecting ionic species in solutions with dissolved salts with different cations and changes of relative humidity in gas phase. This type of nanoparticle nanofibrous scaffold is further demonstrated as a flexible sensor strip for detecting changes in sweating and perspiration for volunteers before and after exercise. The sensitivity of the electrical responses depends on the nature of molecular interactions in the nanocomposite materials. Implications of the findings for potential applications of the flexible nanocomposite scaffolds in developing wearable sweat sensors are also discussed.