Papain-stabilized gold nanoclusters (papain@AuNCs) with a red fluorescence emission at 639 nm have been generated successfully in aqueous solution. The fluorescence of papain@AuNCs could be quenched in the addition of Cu2+. Subsequently, a unique fluorescent probe based on papain@AuNCs-Cu2+ complex has been constructed for sensitive, selective and “turn-on” detection of D-penicillamine (D-pen). The sensing probe has exhibited a remarkable fluorescence enhancement in the presence of D-pen, which can be measured ranging from 30.0 μM to 2.0 mM (r2 = 0.991) with the detection limit of 5.0 μM. Furthermore, the developed assay has been utilized to explore the metabolic process of D-pen in rats after intraperitoneal injection. As far as we concerned, the present study has reported the first analytical method for sensing serum D-pen in real time by using the fluorescent papain@AuNCs-Cu2+ complex. Our strategy paves a way for digging up the biological and clinical application of the fluorescent AuNCs.