Recovering lignin with high β-O-4 content is of prime interest for further high yield depolymerization in low molecular weight phenolic compounds. Pretreatment of lignocellulosic biomass with deep eutectic solvents (DES) was studied to extract this type of tailored lignin from softwood and brewer's spent grains. In this work, choline chloride (ChCl) based DES with two different hydrogen bond donors (HBD) (lactic acid (LA) and Glycerol (Gly)), were investigated at mild temperatures (60 and 80 °C). The influence of DES pretreatment on extracted lignin molecular weight and structural characteristics was analysed. The acidity and density of DES were proved to affect lignin extraction yield and its features. The lignin characteristics (type of interunits, accessibility) were shown to impact their ability to be recovered. Acidic-DES ChCl:LA at 80 °C with woody biomass gave promising results with 78% of lignin extracted exclusively composed of G units with 61% of β-O-4 linkages with narrow molecular weights distribution.