As a representative technology for point‐of‐care testing (POCT), lateral flow immunoassay (LFIA) has been broadly used to detect analytes in many fields. However, its clinical application is severely limited by the unsatisfactory sensitivity, which makes it difficult to obtain accurate results when detecting biomarkers of trace levels, especially in complex matrices. Nanoparticles have been introduced into LFIA for years and become an indispensable part, acting not only as carriers that load and enrich biomolecules, such as antibodies and dyes, but also a miniature platform applied for creative design and construction of nanoprobes. Due to the unique properties at the nanoscale, including the mimetic enzyme activity, the characteristic plasma resonance spectrum and so on, nanomaterials exhibit great potential in the development of novel LFIA and high‐sensitivity detection.