Abstract In this study, a facile non-covalent method was developed for preparing water-soluble graphene with excellent electronic conductivity. Room temperature ionic liquids (ILs) with high ionic conductivity were used for the non-covalent surface functionalization of graphene through π–π stacking interactions. Compared to other ILs used, amino acid ionic liquids (AAILs) were found to be the most effective for improving the dispersion of graphene in water phase. Electrochemical and spectroscopic results confirmed that the obtained AAIL functionalized GR can retain the excellent electronic conductivity of pristine graphene without damaging the graphene lattice. The obtained water-soluble graphene (GR-AAIL) was exemplified to fabricate an electrochemical biosensor using tyrosinase as a model enzyme, and the sensitivity (12,600mAcm−2M−1) of GR-AAIL based biosensor was about 17 times higher than that of graphene oxide and other nanomaterial based biosensor, displaying its unprecedented high sensitivity for biosensing. The detection limit for catechol (one important environmental pollutant) reached as low as 8nM with a response time of 3s and a linear range from 25nM to 11,100nM. The AAIL-GR based biosensor also demonstrated good reproducibility, repeatability, selectivity, long-term stability and high recovery for catechol detection. Amino acid ionic liquid functionalized graphene proves to be a robust and versatile electrochemical biosensing platform for fabricating biosensors with excellent performance.