Atherosclerosis is one of the leading causes of mortality in developed and developing countries. The onset of atherosclerosis development is accompanied by overexpression of several inflammatory chemokines. Neutralization of these chemokines by chemokine-binding agents attenuates atherosclerosis progression. Here, we studied structural binding features of the tick protein Evasin-3 to chemokine (C-X-C motif) ligand 1 (CXCL1). We showed that Evasin-3-bound CXCL1 is unable to activate the CXCR2 receptor, but retains affinity to glycosaminoglycans. This observation was exploited to detect inflammation by visualizing a group of closely related CXC-type chemokines deposited on cell walls in human endothelial cells and murine carotid arteries by a fluorescent Evasin-3 conjugate. This work highlights the applicability of tick-derived chemokine-binding conjugates as a platform for the development of new agents for inflammation imaging.