The preparation of aggregation-induced emission-type copper nanoclusters (CuNCs) capped with polydopamine (PDA) is described. PDA was formed via in situ polymerization of dopamine in the presence of alkaline polyethylenimine. The PDA-capped CuNCs (PDA-CuNCs) exhibit orange fluorescence with maximal emission at 580 nm upon excitation at 340 nm, a storage stability of at least 2 weeks, and a quantum yield (QY) of 2.54% in aqueous solution. The QY is 28-fold higher than that of sole CuNCs. The fluorescence of the PDA-CuNCs is quenched by Fe3+ ion while it is recovered by PO43− due to its stronger affinity for Fe3+. On this basis, a fluorometric phosphate assay was developed that has a 1.5 nM detection limit and a linear range over 0.003–70 μM. The method was satisfactorily applied to the determination of phosphate in local tap water and human sera, and the results agreed well with those obtained by a colorimetric method. In the presence of acid phosphatase (ACP), PO43− is produced by the catalytic hydrolysis of adenosine triphosphate (ACP substrate). Thus, a fluorogenic assay for screening ACP activity was established. Response is linear over the activity range 0.0012–25 U L−1, with a detection limit of 0.001 U L−1 (at S/N = 3). Graphic abstractWe proposed an effective polydopamine-templating strategy for the in situ synthesis of highly emissive and stable CuNCs and demonstrated its use as an ion-driven fluorescence switch for the determination of phosphate and acid phosphatase activity.