A new strategy for preparing sialic acid (SA)‐quenching fluorescent nanoparticles by macromolecular self‐assembly and molecular imprinting technique is suggested. An amphiphilic block copolymer polystyrene‐b‐poly(glycidyl methacrylate) (PS‐b‐PGMA) was designed and synthesized. After functionalization, PS‐b‐PGMA was co‐assembled with the template molecule SA to prepare SA‐imprinted fluorescent nanoparticles, named as nano‐FMIPs. In this process, the shell layers of nanoparticles were photo‐crosslinked upon 365 nm UV light to stabilize the recognition site. The size of the crosslinked shell was found to be increased to 420 ± 5 nm by TEM and DLS measurements. After extraction of SA, recognition cavities complementary in shape, size, and chemical function toward the template SA were left on the surface of nano‐FMIPs, which can selectively rebind the target SA in a mixture of structurally similar compounds. It is found that SA can significantly quench the fluorescence intensity of nano‐FMIPs linearly within a concentration range of 1 × 10−2 ~ 2 × 10−6 mol L−1 and a detection limit of 1.55 × 10−6 mol L−1. This strategy provides a new approach for preparing surface‐imprinted nano‐FMIPs for the quantitative detection of SA, and the nano‐FMIPs may have great potential for biomedical application.