Transient metallochaperone-target protein interactions are essential for intracellular metal trafficking but challenging to study at both the ensemble and the single-molecule level. Here we report using nanovesicle trapping to enable single-molecule fluorescence resonance energy transfer (smFRET) studies of transient interactions between the copper chaperone Hah1 and the fourth metal-binding domain of its target protein, the Wilson disease protein (WDP). We were able to monitor their interactions in real time one event at a time, capture distinct protein interaction intermediates, resolve intermediate interconversion dynamics, and quantify both the interaction kinetics and thermodynamics in the absence of copper. The study exemplifies the ability of nanovesicle trapping in combination with smFRET for studying weak protein interactions and provides insight into how Hah1 and WDP may collaborate to mediate copper transfer inside cells.