Nanomaterials significantly contribute to the development of new solutions to improve consumer products properties. Silver nanoparticles (AgNPs) are one of the most used, and as human exposure to such NPs increases, there is a growing need for analytical methods to identify and quantify nanoparticles present in the environment. Here we designed a detection strategy for AgNPs in seawater using surface-enhanced Raman Scattering (SERS). Three commercial AgNPs coated with polyvinylpyrrolidone (PVP) were used to determine the relative impact of size (PVP-15nmAgNPs and PVP-100nmAgNPs) and aggregation degree (predefined Ag aggregates, PVP-50–80nmAgNPs) on the SERS-based detection method. The study of colloidal stability and dissolution of selected AgNPs into seawater was carried out by dynamic light scattering and UV-vis spectroscopy. We showed that PVP-15nmAgNPs and PVP-100nmAgNPs remained colloidally stable, while PVP-50–80nmAgNPs formed bigger aggregates. We demonstrated that the SERS-based method developed here have the capacity to detect and quantify single and aggregates of AgNPs in seawater. The size had almost no effect on the detection limit (2.15 ± 1.22 mg/L for PVP-15nmAgNPs vs. 1.51 ± 0.71 mg/L for PVP-100nmAgNPs), while aggregation caused an increase of 2.9-fold (6.08 ± 1.21 mg/L). Our results demonstrate the importance of understanding NPs transformation in seawater since this can influence the detection method performance.