Chemiluminescence (CL) immunoassays for simultaneous detection of early acute myocardial infarction (AMI) biomarkers, including copeptin, heart-type fatty acid binding protein (h-FABP), and cardiac troponin I (cTnI), were developed by using Co2+/N-(aminobutyl)-N-(ethylisoluminol) (ABEI) functionalized magnetic carbon composite (Co2+-ABEI-Fe3O4@void@C) as an interface and a three-dimensional microfluidic paper-based device (3D μPAD) as a detection system. For CL immunoassays, Co2+-ABEI-Fe3O4@void@C was assembled with chitosan (CS) and gold nanoparticle-conjugated antibody (Au-Ab) sequentially to form the sensing platform (Co2+-ABEI-Fe3O4@void@C-CS/Au-Ab). In the presence of antigen (Ag), Ag was captured by the sensing interface to form an immunocomplex, leading to an increase in CL intensity due to the catalysis of -COO- existing in Ag. A 3D μPAD with three detection zones for simultaneous detection of copeptin, h-FABP, and cTnI was designed and fabricated to obtain time-resolved CL signals. Three kinds of immunocomplexes formed with copeptin, h-FABP, and cTnI were added to three detection zones of 3D μPAD, respectively. After injecting H2O2, three time-resolved CL signals were generated in one CL detection run by virtue of time-delayed transport of H2O2 to different detection zones. The three time-resolved CL signals were used for the simultaneous determination of copeptin, h-FABP, and cTnI. The detection limit of copeptin, h-FABP, and cTnI was 0.40 pg/mL, 0.32 pg/mL, and 0.50 pg/mL, respectively, which is at least 1 order of magnitude lower than most of the reported immunoassays. The immunoassays could be directly used for the detection of copeptin, h-FABP, and cTnI in human serum samples. The proposed immunoassays are simple, fast, sensitive, and selective, and are of great application potential in early diagnosis and treatment of AMI.