Natural Deep Eutectic Solvents (NADESs) are composed of naturally occurring compounds such as organic acids, amines and sugars; they are biodegradable and have low toxicities. NADESs have gained attention not only as environmentally friendly solvents, but also for their enzyme stabilization effects at high temperatures or osmotic stress. Six betaine-based NADESs were tested regarding their impact on enzymatic activity and thermal stability of a dye-decolourizing peroxidase from the basidiomycete Pleurotus sapidus (PsaPOX). Betaine : sucrose : water (2 : 1 : 10) and betaine : trehalose : water (4 : 1 : 14) had the best impact on the thermostability. Coloured silk was discoloured more efficiently by PsaPOX after addition of either NADES, which indicates a potential as stain-removal agents. The yield of the enzymatic transformation of trans-anethole to the aroma compound p-anisaldehyde increased by a factor of three with both tested NADESs. Overall, it was shown that betaine-based NADESs serve as sustainable media for reactions using PsaPOX and increase temperature stability as well as enzymatic activity. They might thus improve the industrial usability of PsaPOX.