The escalating crisis of nanoplastic pollution in water and food products demands the development of novel methodologies for detection and recycling. Despite various techniques available, surface-enhanced Raman scattering (SERS) is emerging as a highly efficient technique for the trace detection of micro/nanoplastics. However, the development of highly reproducible and stable, flexible SERS substrates that can be used for sensitive detection in environmental medium remains a challenge. Here, we report a fabrication of large-area, three-dimensional (3D), and highly flexible SERS substrate based on porous dendritic Au films onto a flexible indium tin oxide (ITO) substrate via facile, thermal evaporation of Au over the vacuum-compatible deep eutectic solvent (DES)-coated glass substrate and subsequent direct transfer process. The as-fabricated 3D dendritic Au/ITO flexible substrates can be used for ultrasensitive SERS detection of crystal violet (CV) as probe analyte molecules with the limit of detection (LOD) as low as 6.4 × 10-15 M, with good signal reproducibility (RSD of 11.3%). In addition, the substrate showed excellent sensitivity in detecting nanoplastics such as poly(ethylene terephthalate) (200 nm) and polystyrene (100 nm) with LODs reaching up to 0.051 and 8.2 μg/mL, respectively. This work provides a facile approach for the preparation of highly flexible plasmonic substrates, showing great potential for the SERS detection of a variety of environmental pollutants.