<p>A simple, sensitive, and rapid dispersive liquid‒liquid microextraction (DLLME) method is developed for the trace determination of vitamin B₁ HCl (thiamine hydrochloride) in pharmaceutical formulations and seeds. The proposed method relies on the formation of the soluble, highly pigmented dye Prussian blue that is extracted using a mixture of chloroform and acetone; the resulting microorganic droplets are then quantified at a maximum absorption wavelength of 760 nm. All parameters affecting complex formation and DLLME efficiency are systematically optimized. The relationship between absorbance and concentration adhered to Beer’s law, with a linear correlation coefficient (R²) of 0.9992 over a concentration range of 0.03–1.2 μg mL⁻¹. The limit of detection (LOD) and the limit of quantification (LOQ) were determined to be 0.02 μg mL⁻¹ and 0.09 μg mL⁻¹, respectively. Beer’s law was obeyed, with a linear correlation coefficient of 0.9992, in the range of 0.03–0.9 μg mL⁻¹. A concentration of 0.0091 μg mL⁻¹ was determined to be the detection limit, and 0.03 μg mL⁻¹ was found to be the quantification limit. The method was successfully applied and allows for the separation and preconcentration of thiamine hydrochloride at trace levels in biological samples, pharmaceutical formulations and sunflower seed samples.</p>