Bath macrocyclic supramolecules and gold nanoparticies (AuNPs) play important roles in bioanalysis. For the former, molecular recognitions can be carried out adequately; while for the latter, good biocompatibility, high chemical stability, surface functionalization, and facile synthesis make them considerable building blocks for novel hybrid nanomaterials. Some hybrid nanomaterials have been used 2D solution state calorimetric assay. It is a challenge to hybrid AuNPs and macrocyclic supramolecules for the interface-based assays. Here, we report a thionine mediated para-sulfonatocalix[4] arene capped AuNPs (pSC(4)-AuNPs) multilayers signal amplify strategy, which integrates nanoassembly with hybrid macro cyclic supramolecules nanomaterials for the sensitive detection of acetylcholinesterase (AChE) activity, by taking the assay of a neurological diseases marker as an example. In this strategy, the target catalyzing acetylcholine (ATCh) into thiocholine (TCh), while TCh was self-assembled on the gold chip via an Au-S bond. Thionine mediated nanoassemblies of pSC(4)-AuNPs for signal amplification occurring on the electrode interface. As a result, this strategy integrates the advantages of nanotechnology, supramolecular recognition techniques, signal amplification and electrochemical detection and provides a tool for monitoring enzyme. The whole procedure of this novel strategy is similar to hybrid the conventional calorimetric and electrochemical assay, inheriting the usability. But in comparison with calorimetric and electrochemical assay, the performance is greatly improved. The detection limit can be lowered to 2.4 pU/mL, making it possible to detect the target marker sensitively. Therefore, this approach could be further extended to the assays of other targets and may contribute to the development of disease diagnosis and therapy in the future. (C) 2018 Elsevier Ltd. All rights reserved.