An electrochemiluminescent (ECL) aptamer based assay is described for thrombin. It is based on the use of carbon dots (C-dots) placed on polydopamine nanospheres loaded with silver nanoparticles (PDANS@Ag) and with probe DNA (pDNA). The PDANS possess high specific surface and can load a large number of C-dots. The AgNPs, in turn, enhance the ECL emission of the C-dots. Platinum functionalized graphene (Gr-Pt) can connect capture DNA (cDNA). The ECL nanoprobe consisting of PDANS@Ag/C-dots was placed on a glassy carbon electrode modified with Gr-Pt/cDNA/BSA via hybridization between cDNA and pDNA. On applying voltages from -1.8 V to 0 V, a strong ECL signal is generated. If thrombin is added, it will bind to cDNA. This leads to the release of pDNA from the electrode surface and a decrease in ECL intensity. Response to thrombin is linear in the 1.0 fmol·L to 5.0 nmol·L concentration range, with a 0.35 fmol·L detection limit. The assay is stable, repeatable and selective, which demonstrates its clinical applicability. Graphical abstract Carbon dots (C-dots) placed on polydopamine nanospheres loaded with silver nanoparticles (PDANS@Ag) for electrochemiluminescent (ECL) detection of thrombin.