A nanozyme-linked immunosorbent assay is described for cardiac troponin I which is a biomarker for myocardial infarction. The method is based on the use of Pd-Ir nanocubes with excellent peroxidase-like activity. The nanocubes catalyze the oxidization of nonfluorescent o-phenylenediamine (OPD) by H2O2 to form a yellow fluorescent product (oxOPD) with excitation/emission maxima at 400/570 nm. Carbon dots are added as a reference fluorophore. Under the same excitation wavelength, they display blue fluorescence (450 nm). The ELISA uses the Pd-Ir nanocubes as a label for the secondary antibody and OPD as substrate. The ratio of fluorescence intensities at 570 and 450 nm increases in the 1 pg·mL−1 to 1 ng·mL−1 cardiac troponin I concentration range, and the detection limit is 0.31 pg·mL−1. The method was applied to analyze spiked serum samples, and the results compared well with those obtained by a commercial chemiluminescence assay. Graphical abstractSchematic presentation of the ratiometric fluorescence immunoassay for cardiac troponin-I. Pd-Ir nanocubes were employed to fabricate nanozyme-based signal labels for its excellent peroxidase-mimicking activity.