Deep eutectic solvents (DESs) are the next generation of green solvents that are considered for their stability and biocompatibility. This study used 10 different DESs synthesized from choline chloride, alcohols, organic acids and sugars. Red beet betalains were extracted using three conventional solvents and DESs. Characterization experiments of DESs suggested that the electrical conductivity, pH, viscosity, water activity, density and chemical structure were greatly affected by the composition of the hydrogen bond acceptors (HBA) and hydrogen bond donors (HBD). Betacyanin, betaxanthin and total betalain contents ranges were 23.68–702.17, 21.49–467.77, and 45.17–1169.94 mg kg−1, respectively, with choline chloride (ChCl): glucose (Glu) (1:2) giving the highest values. ChCl:Glu was chosen for the optimization process considering the molar ratio (ChC = 1:Glu = 0.75–1.75), water content (15–35%) and temperature (30–60 °C) factors for the central composite design. The optimum conditions were recorded as 1:0.75 molar, 30.83% water content, and 30 °C, respectively. Under optimum conditions, the yields of betalain, betacyanin and betaxanthin were found to be 1192.17 ± 23.63, 738.83 ± 17.87, and 453.34 ± 5.93 mg kg−1, respectively. Bioaccessibility analysis and stability tests were performed on the extracts obtained under optimum conditions. Stability tests revealed that the betalains of red beetroot are less stable in the light than in the dark. Bioaccessibility values for betacyanin, betaxanthin, and betalain were found to be 44.67 ± 1.40, 75.02 ± 1.20, and 56.21 ± 1.33%, respectively. Green extraction of betalains from red beetroot using DES, such as ChCl:Glu, is promising for a strong stabilization and high bioaccessibility of betalains.Graphical