In this work, we report the development of an ultrasensitive sandwich-type electrochemical aptasensor for protein detection. The aptasensor is fabricated by using nitrogen-doped graphene oxide (N-GO) and Au nanoparticles (AuNPs) as sensing substrates, molybdenum disulfide (MoS2) spheres as the hybridization chain reaction (HCR) platform, and thrombin as the model protein. When the hybridization reaction is initiated through two biotinylated hairpin probes, vast horseradish peroxidases are immobilized on the long duplex by the biotin-avidin reaction. An electrochemical-chemical-chemical redox cycling reaction then takes place in the detection system, which contains p-dihydroxybenzene, ferrocene carboxylate and tris(2-carboxyethyl)phosphine. Benefiting from the good conductivity and high specific surface area of N-GO/AuNPs and MoS2 spheres, signal amplification of the HCR and detection system, and excellent selectivity of the aptamer and sandwich-type strategy, the proposed assay shows a wide linear range of 10 fM-0.1 nM towards thrombin with a detection limit of 27 aM (S/N = 3) along with clear distinction from different proteins. The proposed assay is successfully used to detect thrombin in human serum, which would have promising prospects for disease diagnosis and therapy.