We report the first direct, non-invasive experimental evidence of a 2D isotropic-nematic transition for highly anisotropic nanoparticles at liquid-liquid interfaces by using passive fluorescent particle tracking. In order to illustrate the potential of this approach on systems of high real practical and biological relevance, we select as a model anisotropic nanoparticles b-lactoglobulin amyloid fibrils of varying aspect ratios. Upon nanoparticle adsorption at the interface, we follow, in real time and as a function of fibril length and bulk concentration, the development of a 2D nematic phase by studying the anisotropy in probe particle traces. We furthermore demonstrate the long-range nature of the nematic phase by calculating order parameters for the traces as high as 0.8 over 10 2 to 10 3 mm 2 areas. The presented route is independent of the system investigated, and thus these findings open a new, general strategy for the experimental assessment of 2D structural changes at anisotropic fluid-fluid interfaces.