The cobalt compounds have adverse health effect on human and caused damage to the DNA cells, neurological and endocrine systems. Therefore, the separation and determination of cobalt in water and food samples must be considered. In this research, the (2S)-2-amino-3-methyl-3-sulfanylbutanoic acid (penicillamine) as a chelating agent mixed with ionic liquid (OMIM PF6) /acetone and used for extraction of cobalt from 50 mL of water samples by ultra-assisted dispersive liquid-liquid microextraction (USA-DLLME) at pH=6. Based on procedure, the samples were shaked for 5 min (25oC) and after complexation of cobalt ions by thiol and amine group of penicillamine, the ionic liquid phase separated in the bottom of the conical tube by centrifuging for 3.0 min. The upper liquid phase was vacuumed by the auto-sampler and the Co2+ ions back extracted from the ionic liquid/ penicillamine in acidic pH. Finally, the cobalt concentration in remained solution was determined by atom trap flame atomic absorption spectrometry (AT-FAAS). The main parameters such as the sample volume, the penicillamine amount, the ionic liquid amount and the shaking time were optimized. The linear range, the detection limit (LOD) and enrichment factor were obtained 1.5-62 μg L-1, 0.38 μg L-1 and 98.5, respectively (r = 0.9995, RSD%=2.2).