This research describes the spectrophotometric determination of methylene blue (MB) after shaker-assisted liquid-liquid microextraction combined with back-extraction (SALLME-BE). A hydrophobic deep eutectic solvent (DES) was used for extraction of MB from alkaline solutions (0.5 M NaOH). Then, the extracted dye was back-extracted into a hydrochloric acid solution (2 M). Decanoic acid and methyltrioctylammonium bromide were used, respectively as hydrogen bond donor and hydrogen bond acceptor constituents of the hydrophobic DES. The calibration graph was linear in the range of 2–170 ng mL−1. The recovery analysis of wastewater and river water samples verified that this method is practical for environmental monitoring. •Shaker-assisted liquid-liquid microextraction of methylene blue•Application of deep eutectic solvent as environmentally friendly extraction solvent•Spectrophotometric quantification after back-extraction