In this paper, a new microextraction method, named deep eutectic solvent stir bar sorptive extraction (DES−SBSE), is described that utilizes a hydrophobic DES (hDES) as a coating for SBSE. As a model, vitamin D 3 was extracted efficiently by this technique from different real samples before its spectrophotometric determination. A conventional magnet was encapsulated inside a glass bar (1.0 cm × 2 mm) and coated by the hDES consisting of tetrabutylammonium chloride and heptadecanoic acid (mole ratio 1:2). Parameters affecting microextraction were studied and optimized by onevariable-at-a-time, central composite design, and Box−Behnken design methods. Under the optimum conditions, the detection limit of 0.08 μg L −1 could be reached. The linear range of the method was between 0.5 and 1000.0 μg L −1 for the analyte. Precision for intraday repeatability and interday reproducibility of the method was better than 3.1 and 4.2, respectively. A single stir bar could be used for at least 50 successive extractions and the batch-to-batch reproducibility of SB coated with hDES was 4.5%.