In this study, the plasmonic Ag nanoparticles (Ag NPs) were uniformly anchored on the high conductivity NbCT (MXene) nanosheets to construct an Ag/NbCT substrate for surface-enhanced Raman spectroscopy (SERS) detection of polystyrene (PS) nanoplastics. The KI addition (0.15 mol/L), the volume ratio between substrate colloid and nanoplastic suspension (2:1), and the mass ratio of NbCT in substrate (14%) on SERS performance were optimized. The EM hot spots of Ag/NbCT are significantly enlarged and enhanced, elucidated by FDFD simulation. Then, the linear relationship between the PS nanoplastics concentration with three different sizes (50, 300, and 500 nm) and the SERS intensity was obtained (R > 0.976), wherein, the detection limit was as low as 10 mg/mL for PS nanoplastic. Owing to the fingerprint feature, the Ag/NbCT-14% substrate successfully discerns the mixtures from two-component nanoplastics. Meanwhile, it exhibits excellent stability of PS nanoplastics on different detection sites. The recovery rates of PS nanoplastics with different sizes in lake water ranged from 94.74% to 107.29%, with the relative standard deviation (RSD) ranging from 2.88% to 8.30%. Based on this method, the expanded polystyrene (EPS) decomposition behavior was evaluated, and the PS concentrations in four water environments were analyzed. This work will pave the way for the accurate quantitative detection of low concentration of nanoplastics in aquatic environments.