Plastic pollution has reached alarming levels in recent years. While macro- and microplastic pollution are attested and studied since the 1970s, much less is known about the associated nanoscopic fragments. Due to their ability to cross biological barriers and their extended surface area-to-volume ratio, nanoplastics (NPs) are currently considered as one of the major threats for aquatic and terrestrial environments. Therefore, analytical tools are urgently needed to detect and quantify NPs. In this study, a method exploiting the dependence of the fluorescence quantum yield of a probe, namely, 9-(2,2-dicyanovinyl)julolidine (DCVJ), toward its microenvironment was assessed to detect and quantify polystyrene nanoplastics (PSNs). In the presence of PSNs and after excitation at 450 nm, the single-emission band fluorescent molecular rotor (FMR) emission spectrum displays a second peak at 620 nm, which increases with the concentration of PSNs. In pure water, a limit of detection and quantification range of 475–563 μg·L–1 and 1.582–1.875 mg·L–1, respectively, were obtained for 49 nm diameter polystyrene beads (PSB49). The results associated with 100 nm diameter PSNs amount to 518 μg·L–1 and 1.725 mg·L–1. The robustness of the method toward different parameters, the complexity of the matrix, and the PSN characteristics was also assessed. Finally, the method was applied on biological samples. While PSB49 quantification was achieved using radish sprouts at concentrations up to 200 mg·L–1, it was more challenging when handling mussel tissues. This work presents the feasibility to quantify PSNs using DCVJ fluorescence. It paves the way to new perspectives in the challenging field of NPs.