Recent studies on the use of mixed-matrix membrane (MMM) in microextraction methods have considered the use of membrane extraction phase with wide surfaces and high porosity. Here, for the first time, the effect that deep eutectic solvents (DESs) have on the performance of MMM was investigated. The Co-Al-layered double hydroxides mixed matrix membrane (CO-Al-LDH-MMM) was prepared by immobilization of LDH on the pores of the hollow fiber membrane and used as a microextraction device. Deep eutectic solvent via hydrophilic and π-π interactions, aided the dissolution of analyte in the fiber membrane lumen and increases their adsorption onto LDH. Thus, a microextraction method based on the novel LDH-MMM–deep eutectic solvent (LDH-MMM-DES) system was developed and used for the extraction, preconcentration, and quantification of warfarin (as a model analyte) in biological samples. The principal variables that affective influence microextraction efficiency, including the extraction time, type of DES, stirring rate, desorption time, type of eluent solvent, and pH of the sample solution, were screened via Plackett-Burman design and then variables effective optimized by Box- Behnken design. Under the optimum conditions obtained, the developed method showed a good working range (R2 ≥ 0.9951). The limit of detection (LOD) and the limit of quantification (LOQ) ranged from 0.03 μg L 1 (water) to 0.30 μg L 1 (human plasma sample) and from 0.099 μg L 1 (water) to 0.999 μg L 1 (human plasma sample) variables, respectively. The intraday precision was in the range of 2.5–5.6. In order to experiment with the precision and applicability of the Co-Al LDH-MMM DES method, spiking recovery studies were performed in biological samples (urine and humane plasma samples). The recovery results for urine and plasma samples were calculated as 90–91% and 88–93 %, respectively. The results confirmed that the presented method would be promising for the analysis of warfarin in biological sample
