In this work, a cleancap-regulated aggregation-induced emission (AIE) strategy based on copper nanoclusters (CuNCs) was developed with stepwise recognition for highly specific analysis of the enzyme. The dissolved CuNCs with AIE characteristics in alkaline solution were prepared by using p-mercaptophenylboronic acid as the reducing agent and the stabilizing ligand. The prepared CuNCs can specifically conjugate with glucose (Glu) to connect with each other via the rapid boronate esters formation between boronic acids of CuNCs and a pair of cis-diols on Glu. The cleancap-regulated AIE strategy was further identified by modification of CuNCs with d-glucose 6-phosphate (P-Glu) as the capper and substrate. Introduction of alkaline phosphatase to the P-Glu/CuNCs complex can induce the cleavage of phosphate group to activate the 5,6-diol of Glu on the CuNCs. The decapped complexes could be aggregated through further conjugation between 5,6-diol and boronic acid of two CuNCs, resulting in strong red AIE luminescence. The dual recognitions of enzymatic cleavage and cis-diols/boronic acid conjugation endow the designed method with highly specific detection and cell imaging of enzymatic activity. The cleancap-regulated AIE strategy provides a universal tool for regulation of AIE phenomenon in trace analysis.