This research reports an eco-friendly oat protein extraction method using choline chloride (ChCl)–butanediol deep eutectic solvents (DESs) and DES/water binary mixtures. Eighteen formulas consisting of ChCl with butanediol isomer (1,2-butanediol, 1,4-butanediol, or 2, 3-butanediol) in the absence or presence of water at different molar ratio were examined for the synthesis of DESs and binary mixtures. We found that 6 formulae can be fabricated at a ChCl–butanediol molar ratio of 1:3 independent of water presence, and that they had better oat extraction performance with 90 min extraction at 80 °C. Under the optimal conditions, we further assessed the impact of butanediol isomers and water addition on the structure characteristics and functionalities of extracted oat protein. The results suggested that oat protein extracted by ChCl−1,4-butanediol/water binary mixture had highest protein content, solubility, foaming capacity and stability. Our pilot findings provide crucial support to our hypothesis that the isomer of hydrogen bond donor is critically involved in the properties of oat protein the DES and the binary mixtures extracted, which pave the way for the future application of DES in protein extraction.