In this work, I present the application of single-molecule imaging to systems biology and discuss the relevant technical issues within this context. Imaging single molecules has made it possible to visualize individual molecules at work in living cells. This continuously improving technique allows the measurement of non-invasively quantitative parameters of intracellular reactions, such as the number of molecules, reaction rate constants and diffusion coefficients with spatial distributions and temporal fluctuations. This detailed information about unitary intracellular reactions is essential for constructing quantitative models of reaction networks that provide a systems-level understanding of the mechanisms by which various cellular behaviors are emerging.