A label-free, sensitive and simple method to detect protein kinase based on the selective aggregation of phosphorylated peptide-gold nanoclusters (peptide-AuNCs) triggered by Zr4+ ion coordination is developed. The AuNCs were synthesized by peptide without any strong reducing agents, which prevent peptides from being disrupted. Under optimal conditions, a linear relationship between the decreased PL intensity of peptide-AuNCs and the concentration of casein kinase II (CK2) in the range of 0.08–2.0unitmL−1 with a detection limit of 0.027unitmL−1 (3σ) was obtained. The feasibility of this AuNCs-based sensor was further demonstrated by the assessment of kinase inhibition by ellagic acid, 5,6-dichlorobenzimidazole-1-β-d-ribofuranoside, emodin, and quercetin in human serum. As expected, the PL intensity increased with increasing inhibitor efficiency in the presence of inhibitors. The IC50 value (inhibitor concentration producing 50% inhibition) for ellagic acid was estimated to be 0.045μM. With more sophisticated design of the peptide substrate sequences, the detection of other enzymes will be realized. With characteristics of homogeneous, facile, universal, label-free, and applicable for kinase assay, the proposed sensor provides potential application in kinase-related biochemical fundamental research and inhibitor screening. •We reported a novel fluorescence assay base on peptide-AuNCs for sensitive analysis of protein kinase CK2 activity.•The peptide-AuNCs was synthesized in situ without any coupling and reducing agents.•Sensitive detection of CK2 based on the aggregation quenching mechanism.•Highly sensitive detection of CK2 activity and inhibition was achieved.