Proteins generally tend to aggregate with less desirable properties in numerous solvents, which is one of the major challenges in the development of solvents for functional proteins. This work aims to utilize fluorescence spectroscopy and small angle X-ray scattering (SAXS) to understand the effects of ionic liquids (ILs) on the fluorescence and aggregation behavior of superfolder green fluorescent protein (sfGFP). The studied ILs consisted of four different anions coupled with primary, tertiary and quaternary ammonium cations. The results show that the chromophore fluorescence was generally maintained in 1 mol% IL-water mixtures, then decreased with increasing IL concentration. We primarily employed the pseudoradius of gyration (pseudo-R-g) to evaluate sfGFP aggregation. The sfGFP was less aggregated with nitrate-based ILs compared to in buffer, and more aggregated in the mesylate-based ILs. Further, we show that the polyol additives of glycerol and glucose in IL-water mixtures slightly decreased the sfGFP propensity to aggregate. Size-exclusion chromatography (SEC)-SAXS was used to characterize the monomeric sfGFP in ethylammonium nitrate (EAN) and triethylammonium mesylate (TEAMs)-water mixtures. The presence of 1 mol% TEAMs maintained the sfGFP fluorescence, promoted the compact structure, but slightly increased the amount of large aggregates, which contrasted with that of EAN. (C) 2021 Elsevier Inc. All rights reserved.