In this work, we outline a signal amplification strategy using the coordination chemistry between Fe3+ and poly(glutamic acid) (PGA) for biosensing applications. The theoretical calculation based on density functional theory shows that PGA has a much higher binding affinity with Fe3+ than the other metal ions. Guided by this rationale, we prepare a PGA-mediated signal probe through conjugating PGA onto polystyrene (PS) nanoparticles to form a brushlike nanostructure for Fe3+ coordination. This PGA-PS brush (PPB) has a large loading capacity of Fe3+ with a number of 1.92 × 108 Fe atoms per nanoparticle that greatly amplifies the signals for assays in an enzyme-free way. Combined with ferrozine coloration-based readout, this PPB-mediated amplification is further applied for the enzyme-free immunoassay that shows an ultrahigh sensitivity for detection of microcystins-LR (12 pg/mL), a 5-fold enhancement compared with that of traditional enzyme-linked immunosorbent assay (ELISA) (60 pg/mL). In addition, the good stability, rapid response, and long shelf life make this enzyme-free amplification strategy a promising platform for point-of-care biosensing applications.