Cytochrome c, an iron containing metalloprotein in the mitochondria of the cells with an oxide/redox property, plays key role in the cell apoptotic pathway. In this study, the interaction of silver nanoparticles (AgNPs) with cytochrome c (Cyt c) was investigated by using a combination of spectroscopic, imaging and thermodynamic techniques, including dynamic light scattering (DLS), ultraviolet–visible (UV–vis) spectroscopy, transmission electron microscopy (TEM), fluorescence spectroscopy, near and far circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC). DLS and UV–vis analysis evidenced the formation of surface complexes of Cyt c on AgNPs. The saturation of surface coverage of AgNPs was observed at 4.36 Cyt c molecules per nm2 of A