Benzotriazole (BTRs) and benzothiazole (BTHs) derivatives have been classified as high production volume pollutants of emerging concern. The present work describes a rapid and simple process using an eco-friendly deep eutectic solvent (DES) based-ultrasound-assisted liquid-liquid microextraction (DES-UALLME) technique to effectively extract five BTRs and four BTHs in human urine samples, and then applying ultrahigh-performance liquid chromatography and electrospray ionization (+)-quadrupole time-of-flight mass spectrometry (UHPLC-ESI(+)-QTOF-MS) for their detection and quantification. DESs are a group of novel “green” solvents, and their applications in sample pretreatment are appropriate for the requirements for green chemistry, environmental protection and sustainable development. Furthermore, to overcome the challenges related to different experimental conditions, multivariate experimental design approaches conducted by means of a multilevel categorical design and a Box-Behnken Design were applied to screen and optimize parameters that have significant influences on the extraction efficiency of DES-UALLME. After optimization, the method was validated and shown to possess low limits of quantitation (LOQs; 0.4 − 9 ng mL−1), high precision (3–12%), and high accuracy (mean spiked recoveries; 80–101%). The developed method was then successfully applied for the analysis of BTRs and BTHs in human urine samples. Interestingly, 5,6-dimethyl-1H-benzotriazole (XTR) was detected in almost all of the urine samples, which correlates with its high production and widely applications in industry processes and consumer products in Taiwan. These target analytes could potentially be used as biomarkers to assess exposure of BTRs and BTHs in biomonitoring programs and studies.