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 β-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 102 to 103μm2 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.