In this work conventional Uv-Vis spectroscopy was used for trace analysis of Dysprosium (III). In order to improve selectivity and detection limit of method, ultrasound-assisted dispersive liquid-liquid micro-extraction (DLLME) was applied for extraction of Dy(III) from aqueous sample. The method was based on the formation of Dy(III)–alpha-benzoin oxime complex and its extraction in to chloroform using a DLLME technique. It was found that the crucial factor for the complex formation is an alkaline alcoholic medium. The effective parameters on the separation and determination process such as pH, ligand concentration, amount and type of dispersive, and extracting solvent were investigated and optimized. Under the optimized conditions, the calibration graph was linear in the ranges of 6.1×10-7–6.8×10-4mol L-1 with the detection limit of 3×10-7mol L-1. The pre-concentration of 20mL of sample gave enhancement factor of 70. The proposed method was applied for determination of Dy(III) ion in spiked urine and water samples.