In this paper, a high-efficiency electrochemiluminescence (ECL) ternary immune-system was firstly constructed for ultrasensitive assay of alpha-fetoprotein (AFP) based on the Ti, N/Mg co-doped carbon dots (Ti,Mg@N-CDs) as brand-new nanocomposite emitter, Poly(DVB-Co-PBA) as novel AFP-signal reporter and coreaction accelerator. A fluorescent microspheres Poly(DVB-Co-PBA), synthesizing based on 4-vinylphenylboronic acid (4-MPBA) and divinylbenzene (DVB), was used as a signal indicator-probe via the specific carbohydrate-AFP interaction to fabricate a dual-recognition label-free immune-co-chemical biosensor. The as-prepared Ti,Mg@N-CDs exhibited high ECL emission of 3.7-folds compared with CDs. Moreover, the large surface area, promising biocompatibility and nitrogen-rich functional group bearing on the nanohybrid was helpful for the antibody adsorption immobilization of AntiAFP/Ti,Mg@N-CDs/GCE. The successful synthesis and enhancement mechanism of the nanomaterials were also characterized through a serious of techniques including HR-TEM, SEM, EDS, FT-IR, UV–vis, PL and CV. With an immune-co-chemical ECL-assay format, target and signal reporter-probe (Poly(DVB-Co-PBA)) were introduced in sequence via specific reaction. Based on multiple signal amplification and dual recognition strategy, a largely linear detection ranges from 2.25 × 10−6 to 2250 ng mL-1 with a low detection limit (1.55 × 10-7 ng mL-1) were obtained via the resultant ECL ternary (Poly(DVB-Co-PBA)-AFP/AntiAFP/Ti,Mg@N-CDs/GCE) system. This proposed new method was applied to detect human serum samples with satisfactory results and acceptable recovery (96.04 % ∼ 111.22 %), indicating a potential application in clinical monitoring of disease biomarkers.