Herein, we developed a platinum-copper nano-enzyme-linked immunosorbent assay (NLISA) based split diagnostic platform for the ultrasensitive detection of cardiac troponin I (cTnI). The PtCu nanozyme synthesized by one-pot synthesis exhibited ultra-high peroxidase-like activity (35.17 U mg−1), which was about 4.5 times higher than that of the unmodified Pt nanozyme (8.83 U mg−1). Due to the efficient peroxidase-like activity of the copper-platinum complexed nanozyme, transduction and sequential amplification of cTnI biological signals were achieved in combination with a liposome-embedded amplification strategy. The encapsulation efficiency was calculated by introducing a liposomal bilayer model, which showed that the introduction of a single liposomal molecule could amplify the signal up to 870-fold, thus promising a high sensitivity test. Notably, the dynamic response of cTnI was in the range of 0.1–5000 pg mL−1 with an ultra-low detection limit (0.048 pg mL−1). The developed NLISA analysis system provides a new way to discover efficient and sensitive alternatives to ELISA kits, which can meet the practical needs of community healthcare testing conditions and rapid testing in hospitals.