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Aminopeptidase N (APN) is an important ectopeptidase involved in many physiological functions e.g. tumor angiogenesis, and the detection of APN activity thus facilitates the early diagnosis and elucidation of tumor development. Herein we propose a one-pot and one-step colorimetric sensing method for detecting APN activity based on a peptide functionalized gold nanoparticles/cucurbit[8]uril (pep-AuNPs/CB[8]) supramolecular structure. In principle, a peptide that comprises both the recognition sequence of APN and binding motif of CB[8] is first functionalized onto the surface of AuNPs. Because CB[8] can interact with two binding motifs simultaneously, the assembly of pep-AuNPs and CB[8] consequently take place, leading to the formation of pep-AuNPs/CB[8] supramolecular structure. However, in the presence of APN, the peptide will be hydrolyzed by APN, resulting in the breach of binding motifs of CB[8] as well as the prevention of the formation of pep-AuNPs/CB[8] supramolecular structure. In this way, the activity of APN can be determined through the absorbance changes based on the assembly/disassembly of AuNPs. Under optimized conditions, the detection of APN activity is achieved in a linear range from 5 μg/mL to 15 μg/mL with a detection limit of 0.42 μg/mL, which is also available directly in serum samples. Because of the simplicity and the cost-efficient materials, the method is competent for practical applications. [ABSTRACT FROM AUTHOR]