This study investigates the potential of thermoresponsive hydrogels as innovative substrates for future in vitro diagnostic (IVD) applications using AVAC technology, developed and patented by the Mecwins biomedical company. In order to convert the hydrogel in a substrate compatible with AVAC technology, the following prerequisites were established: (1) the hydrogel layer needs to be permeable to gold nanoparticles (AuNPs), and (2) the optical properties of the hydrogel should not interfere with the detection of AuNPs with AVAC technology. These two key aspects are evaluated in this work. A silicon substrate (Sil) was coated with a layer of a thermosensitive hydrogel (TSH) based on poly(N-isopropylacrylamide-co-N,N'-methylene bis(acrylamide) (PNIPAAm-co-MBA). The TSH offers the advantage of easy modulation of its porosity through cross-linker adjustments, crucial for the plasmonic nanoparticle (NP) permeation. The platforms, denominated as (Sil)-g-(PNIPAAm-co-MBA), were fabricated by changing the cross-linker concentrations and exploring three deposition methods: drop casting (DC), spin coating (SC), and 3D printing (3D); the DC approach resulted in a very homogeneous and thin hydrogel layer, very suitable for the final application. Furthermore, after physical-chemical characterization, the TSH demonstrated its functionality in regulating nanoparticle absorption, and AVAC technology’s capability to precisely identify such NPs through the hydrogel matrix was validated. The proposed hydrogel platform fulfills the initial requirements, opening the possibility for employing these hydrogels as dynamic substrates in sandwich immunoassay devices. The next step in the development of the hydrogel substrate would be its functionalization with biorecognition groups to allow for biomarker detection. By leveraging their enhanced capture efficiency and the ability to manipulate particle flow thermally, we anticipate a significant advancement in diagnostic methodologies, combining the spatial benefits of three-dimensional hydrogel structures with the precision of AVAC’s digital detection.