Detecting and quantifying molecular species at trace levels is crucial for understanding fundamental processes in chemistry and biology; however, the complexity of real-life samples renders their direct analysis extremely challenging. Here, we demonstrate a surface-enhanced Raman spectroscopic approach for the direct on-site detection and quantification of trace analytes in real-life samples. The key to our approach is the creation of multifunctional plasmonic films via interfacial self-assembly using colloidal Au@Prussian blue (PB) supraparticles as the functional building block. The supraparticles contain 3-dimensional hot-spots that give rise to strong plasmonic near-field enhancement, while the PB shell acts simultaneously as a molecular sieve and internal standard to induce selective analyte adsorption and to calibrate signal fluctuations. This enables direct identification and quantitation of a range of weakly adsorbing targets in biological and environmental samples using a portable Raman spectrometer, which paves the way for rapid on-site chemical analysis in important applications, including therapeutic drug monitoring and environmental analysis.