Protein-protein and protein-DNA interactions play critical roles in biological systems, and these interactions often involve complex mechanisms and inhomogeneous dynamics. Single-molecule spectroscopy is a powerful and complimentary approach to decipher such spatially and temporally inhomogeneous protein interaction systems, providing new information that are not obtainable from static structure analyses, thermodynamics characterization, and ensemble-averaged measurements. To illustrate the single-molecule spectroscopy and imaging technology and their applications on studying protein-ligand interactions, this chapter focuses on discussing two recent single-molecule spectroscopy studies on protein-protein interaction in cell signaling process and on protein-DNA interactions in DNA damage recognition process.