Herein, a green dispersive liquid-liquid microextraction (DLLME) based on hydrophilic and hydrophobic deep eutectic solvents (DESs) was developed for the enrichment of bicalutamide (BIC) from water and spiked human plasma. Six hydrophobic and three hydrophilic DESs were synthesized and evaluated. The hydrophilic DES, composed of tetrabutylammonium bromide and acetic acid in a molar ratio of 1:2, was used as a dispersive solvent. Moreover, the hydrophobic DES, composed of borneol and capric acid in a molar ratio of 1:2, was applied as an extracting solvent. Significant factors affecting the extraction efficiency were investigated through the one-factor-at-a-time (OFAT) strategy. The optimized method was validated in terms of linearity, limits of detection and quantitation, accuracy, precision, and matrix effect. Good linearity with an excellent correlation coefficient (r2 > 0.9999 and 0.9987) was achieved as well as low limits of detection of 0.022 and 0.048 μg/mL and preconcentration up to 53.2 and 92.1 folds for water and plasma, respectively. The proposed DLLME method was coupled with spectrophotometric detection and applied for the analysis of BIC in river water and spiked human plasma with satisfactory recoveries (92.54–99.39%). Finally, the greenness of the developed procedure was evaluated using Complex Green Analytical Procedure Index (ComplexGAPI) and Analytical GREEnness (AGREEprep) tools, exhibiting excellent greenness in comparison to the previously reported methods.