A hydrophobic deep eutectic solvent-based liquid-phase microextraction combined with UV-Vis spectrophotometry was presented for the determination of selenium in eggs and chicken tissue for the first time. Selenium is commonly used in the poultry industry as an additive in broiler feed and its determination in chicken products is important analytical task. In this study, deep eutectic solvents based on terpenoids (thymol and menthol) and fatty acids were investigated as extraction solvents for the sensitive determination of trace selenium amounts by UV-Vis spectrophotometry. The procedure involved microwave digestion of food sample, formation of yellow compound of piazselenol in the presence of selenium (IV) and 3,3′-diaminobenzidine followed by piazselenol microextraction. The quantification of selenium in deep eutectic solvent phase was carried out in a flow cell of a portable UV-Vis spectrometer. The sensitivity was improved by about 300 folds when compared with the UV-Vis spectrophotometry without analyte preconcentration. Deep eutectic solvent based on thymol and menthol provided suitable conditions for analyte detection. The analytical parameters for the quantitative microextraction of analyte were investigated and optimized. Validation of the procedure was performed. The linearity range was 7–150 µg kg−1 (0.14–3 µg L−1) and the limit of detection and limit of quantification were 2 µg kg−1 (0.04 µg L−1) and 7 µg kg−1 (0.14 µg L−1). The proposed procedure was successfully applied to analysis of real eggs and chicken tissue samples. The procedure does not require sophisticated and expensive analytical instrumentation and can be found application in food quality control laboratories.