An innovative deep eutectic solvent with ultrasound-assisted based dispersive liquid-liquid microextraction method was first time develop for separation and preconcentration of trivalent chromium in environmental water samples. The `deep eutectic solvent with ultrasound-assisted based dispersive liquid-liquid microextraction method was prepared from acetamide and zinc chloride at different molar ratio. The complexing agent calmagite was used for complex formation with trivalent chromium in the range of 8.0–10 optimum pH values. After that complex of trivalent chromium with calmagite was entrap by using deep eutectic solvent. Extraction efficiency was increase by adding tetrahydrofuran into the extraction unit. Extracted and pre-concentrated trivalent chromium contents from water determined by using graphite furnace atomic absorption spectrometry. Effected extraction efficiency parameters are pH, concentration of calmagite, volume of deep eutectic solvent, volume of tetrahydrofuran, molar ratio of the deep eutectic solvent and extraction time were optimized. The deep eutectic solvent with ultrasound-assisted based dispersive liquid-liquid microextraction method has excellent analytical figure of merits such as enrichment factor and limit of detection 85.1 and 6.0 ng L−1, respectively. Accuracy of the developed method was checked by the certified reference materials of water (TM-28.3) with percentage recovery >98%. The proposed method was successfully applied for determination of trivalent chromium in different natural water samples. •An inventic DES-UA-DLLME method was developed for chromium extraction.•The calmagite was used for complex formation with chromium.•DES was formed by zinc chloride with acetamide at 1:2 M ratio.•DES-UA-DLLME method was applied to chromium in water samples by GFAAS.•Low detection limit and short extraction time were observed.