Microarrays are particular biosensors with multiple grafted probes that are generally used for parallel and simultaneous detection of various targets. In this study, we use microarrays with aptamer probes in order to follow up the different biomolecular interactions of a single enzyme, the thrombin protein, involved in the complex coagulation cascade. More precisely, thanks to the label-free Surface Plasmon Resonance imaging, we are able to monitor in real-time an important step in the firing of the coagulation cascade in situ: the enzymatic transformation of prothrombin into thrombin, catalyzed by the factor Xa. We are also able to appraise the influence of other biochemical factors and their corresponding inhibiting or enhancing behaviors on thrombin activation. Our study not only opens the door for the development of a complete microarray-based platform for the whole coagulation cascade analysis, but also for novel drug screening assays in pharmacology.