Nanoplastics are emerging contaminants in aquatic environments, raising concerns due to their persistence and potential biological impacts. Reliable, rapid, and timely detection methods are therefore needed to address these concerns. Conventional analytical techniques, however, are time-consuming and require specialized instrumentation, limiting routine monitoring of nanoplastics in environmental waters. In this study, we developed a fluorescence-based lateral flow assay (LFA) for the on-site detection of nanoplastics in environmental waters. The assay employed 1-pyrenebutyric acid N-hydroxysuccinimidyl ester (PBN) as a fluorescent probe and polyethyleneimine (PEI) as a capture reagent. The LFA successfully detected five polymer types within 10 min, with limits of detection (LODs) ranging from 9.3 to 163.9 μg/L, depending on polymer type and particle size. The selectivity of the assay was maintained in the presence of common environmental interferents, including bacteria and organic matter. Furthermore, successful detection of nanoplastics in river water and seawater demonstrated its applicability to complex environmental matrices. These results demonstrate the potential of the fluorescence-based LFA as a rapid, portable platform for on-site monitoring of nanoplastics in both freshwater and marine environments.