Prior to FAAS determination, a green, simple, and validated ultrasound-assisted ionic liquid-based dispersive liquid-liquid microextraction technique (UA-IL-DLLME) was developed for preconcentration and separation of cobalt (Co(II)) and nickel (Ni(II)) ions in various environmental and biological samples. The suggested method uses an ionic liquid (IL) (1-hexyl-3-methylimidazolium tris(pentafluoroethyl)trifluorophosphate [HMIM][FAP]) as an extraction solvent for Co(II) and Ni(II) ions following complexation with 3-(2-hydroxy-5-methyl-1-ylazo)-1,2,4-triazole (HMAT) at pH 8. The effects of several analytical factors on microextraction performance were examined. Under ideal conditions, the calibration curves were linear in the ranges of 1–400 and 1-300 μg L−1, with limits of detection of 0.30 and 0.28 μg L−1 for Co(II) and Ni(II), respectively. The preconcentration factor was 100. The analyte recovery rates ranged from 96to 102 percent. Furthermore, for Co(II) and Ni(II), the relative standard deviation (RSD%) for intra-day (1.20 and 1.50%) and inter-day (1.60 and 1.80%) as precision, respectively. The proposed preconcentration approach was tested using certified reference materials (SRM 1570A spinach leaves and TMDA-52.3 fortified water). The suggested UA-IL-DLLME method was successfully used to preconcentrate and determine the concentration of Co(II) and Ni(II) ions in a variety of real environmental (water, juice, and food) and biological (hair) samples, producing satisfactory results.