In this study, Pd@Au nanocubes supported β-cyclodextrins functionalized three-dimensional porous graphene (CDs-3D-PG-Pd@Au NCs) was synthesized using a facile method. β-cyclodextrins (CDs) were beneficial in improving the dispersibility of three-dimensional porous graphene (3D-PG) and displayed good capture capability towards secondary antibodies (Ab2). Moreover, large amounts of Pd@Au NCs could load on the CDs-3D-PG, which effectively improved the electrochemical signals. The obtained CDs-3D-PG-Pd@Au NCs composite was utilized as signal amplification labels. Furthermore, Au nanoparticles (AuNPs) and thionine (Th) decorated on amino-functionalized microporous carbon spheres (AuNPs-FMCS-Th) as sensor platforms, which not only effectively immobilized primary antibodies (Ab1) by interacting with Au-NH2, but also accelerated the electron transfer process on the electrode surface using the mediated effect of Th, resulted in further amplification of the signal response. The morphology and composition of the as-prepared nanomaterials were characterized using scanning electron microscopy (SEM), UV-vis spectroscopy, Raman spectroscopy and transmission electron microscopy (TEM). Cyclic voltammetry (CV) and amperometric i-t methods were used to investigate the electrocatalytic reduction of H2O2 by CDs-3D-PG-Pd@Au NCs using electron mediation of Th. Under optimal conditions, the proposed immunosensor exhibited high selectivity, acceptable stability and good reproducibility for the detection of cardiac troponin I (cTnI) with a low detection limit of 33.3 fg mL-1. Importantly, satisfactory results were obtained for analysing real serum samples, indicating that the designed method could provide an effective strategy in clinical research.