Magnetic fluorescent nanoparticles have demonstrated notable advantages as multifunctional immunoassays in lateral flow biosensor (LFB), contributing to the enhancement of both the flexibility and practicability of LFB. Herein, we synthesized flower-shaped magnetic fluorescent Nanoparticles (FMNP@QDs) with heightened fluorescence emission. The distinctive core/shell architecture of FMNP@QDs comprised the high saturation magnetization core encapsulated by oleic acid-modified Fe3O4, the flower-shaped shell made up of polymer colloids, and quantum dots (QDs) uniformly distributed on the outside. After surface modified with PEG-COOH and conjugate with cardiac troponin I (cTnI) antibodies, the FMNP@QDs were successfully applied in LFB for highly sensitive cTnI detection in samples. The limit of detection (LOD) of the proposed LFB for cTnI was 0.0097 ng/mL under fluorescent mode and 0.026 ng/mL under magnetic mode. A high level of concordance was observed between the FMNP@QDs based fluorescent LFB and the commercial CLIA method. The results indicated that the FMNP@QDs based LFB has a substantial potential for point-of-care test (POCT) technology, owing to its simple operating procedures, affordability, and enhanced sensitivity.