Plasminogen (Plg) plays a key role in fibrinolysis, wound healing, tissue remodelling and cell migration; it is a 7-domain protein consisting of a Pan domain, 5 kringle domains and a serine protease domain and binds to a range of different targets including cell surface receptors. Annexin A2/S100A10 heterotetramer is one of such receptors, and it plays a key role in promoting tumour cell invasion. It has been established that the mechanism through which Plg interacts with Annexin A2/S100A10 heterotetramer is via the lysine binding sites of Plg and S100A10, yet, the detailed molecular interaction remains ambiguous. Here, we use Surface Plasmon Resonance (SPR) to map the key domains of Plg that mediates its interaction with S100A10 using the individual domains of Plg as well as 2-5 domains in combination. We show that kringle-5 and serine-protease (SP) domain are both critical for S100A10-binding. Further, to our surprise, the lysine-analogue EACA does not completely abolish binding of Plg to S100A10, providing further evidence for lysine-independent interactions. Together, these results indicate that the kringle-5 and SP domains predominantly mediate the binding of Plg to S100A10. These studies provide potential insight into mechanisms of Plg activation at the cell surface and the subsequent enhancement of metastasis.