BackgroundAntibiotics residues can accelerate the growth of drug-resistant bacteria and harm the ecological environment. Under the effect of enrichment and biomagnification, the emergence of drug-resistant pathogenic bacteria may eventually lead to humans being ineffective to drugs in the face of bacterial or fungal disease infections in the future. It is urgent to develop an efficient separation medium and analytical method for simultaneous extraction and determination of antibiotics in the water environment. ResultsThis work doped 2,6-Di-O-methyl-β-cyclodextrin, randomly methyl-β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin with thymol:fatty acid respectively to construct non-covalent interaction-dominated pH-responsive ternary supramolecular deep eutectic solvents (SUPRADESs), which can undergo a hydrophilic/hydrophobic transition with aqueous phase to achieve an efficient microextraction. Semi-empirical method illustrated that SUPRADESs have a wide range of hydrogen bond receptor sites. We developed a SUPRADES-based analytical method combined with liquid chromatography-triple quadrupole mass spectrometry for the extraction and determination of trace quinolones and sulfonamides in wastewater. The overall limits of detection of the method were 0.0021–0.0334 ng mL−1 and the limits of quantification were 0.0073–0.1114 ng mL−1. The linearity maintained good in the spiked level of 0.01–100 ng mL−1 (R2 > 0.99). The overall enrichment factors of the method were 157–201 with lower standard deviations (≤8.7). SignificanceThe method gave an extraction recovery of 70.1–115.3 % for 28 antibiotics in livestock farming wastewater samples from Zhejiang, China, at trace levels (minimum 0.5 ng mL−1). The results demonstrated that inducing the phase transition between SUPRADES and aqueous phase by adjusting pH for extraction is a novel and efficient pretreatment strategy. To our knowledge, this is the first application of cyclodextrin-based ternary SUPRADESs with pH-responsive reversible hydrophobicity-hydrophilicity transition behavior in wastewater analysis.