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In this work, we designed a process to assemble gold nanoparticles onto a three-dimensional (3D) polymer surface, which can then be monitored using surface-enhanced Raman scattering (SERS). This work is the first demonstration of the assembly of gold nanoparticles on a filter film and in situ measurement with Raman spectroscopy. Herein, a polyhexamethylene adipamide (Nylon66) film embedded in the organic filter film was used as a template to fabricate a tunable SERS-active substrate. A "hotspot"-rich gold-nanoparticle-decorated polymer substrate for SERS was prepared; this substrate exhibited high sensitivity in trace detection of targets. The study was conducted using 4-mercaptobenzoic acid as a probe molecule with the aim of comparing the scattering efficiency and the homogeneity of the Raman signal on selected substrates. In addition, we used the gold-decorated polymer film to detect a biotin-avidin complex. The most powerful advantage of the proposed microanalytical device is the in situ SERS application. The 3D nanoporous structures described in this work hold strong potential for use in various applications such as environmental monitoring and biomolecule detection.