The exploration of insulin detection is of great significance for achieving diabetes monitoring and treatment. In our study, protamine-encapsulated DNA copper nanoclusters (Prot@DNACuNCs) were synthesized as a novel fluorescent nanoprobe for detecting insulin, a unique biomarker of diabetes. In our results, we found that our synthesized Prot@DNACuNC is highly sensitive toward insulin in a fluorescent detection assay. Further, we focused on fluorescence-based sensing of insulin secretion in response to the function of the pancreatic β-TC-6 cell line under different external glucose concentrations. We found that Prot@DNACuNC is highly sensitive to a glucose-responsive insulin-releasing system via pancreatic β-TC-6 cells. In the mechanism study, we have found that upon contact with insulin, Prot@DNACuNCs firmly bind to it, leading to fluorescence quenching due to: (i) a conformational transition of insulin from α-helical to β-sheet structures, an indicator of its aggregation; and (ii) morphological changes in the nanoprobe. Leveraging the advantages of protamine encapsulation and the unique properties of DNA-templated copper nanoclusters (DNACuNCs), including small size, excellent photostability, large surface area, and good biocompatibility, the developed Prot@DNACuNCs sensing platform serves as a robust fluorescent sensor for insulin detection. Further, this sensor platform showed an ultralow limit of detection (LOD) of 9x10^-9 mol L^-1. Selectivity assays showed minimal interference from other biologically relevant proteins, including HSA, BSA, lysozyme, and haemoglobin. This selective protein encapsulation of DNACuNCs would provide a potential strategy for constructing future nanoprobes by exploiting other encapsulating molecules for the detection of other targets.