A series of supramolecular assemblies based on multi-charged calixarene (SC4A), bis(p-calixarene) (BSC4A) and pillararene (CP5A) modified gold nanoparticles (AuNP) was constructed to realize colorimetric sensing of both succinylcholine (SuCh) and butyrylcholinesterase (BChE). With the high binding affinity of BSC4A and CP5A towards SuCh, BSC4A-AuNPs and CP5A-AuNPs could assemble with micromolar level SuCh as SuCh-BSC4A/CP5A-AuNPs. More interestingly, the enzymatic hydrolysis of SuCh by BChE could lead to the disassembly of SuCh-BSC4A/CP5A-AuNPs and provide a sensitive time-dependent color change from blue to red which could be observed by the naked eye and used to monitor BChE activity. As BChE activity is an important biomarker for diseases and poor health conditions, this novel supramolecular tandem colorimetric sensing strategy may have potential use for early diagnosis of diseases.