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In this study, we investigated the process of preconcentrate and determine trace amounts of Auramine O (AO) and methylene blue (MB) dyes in environmental water samples. For this purpose, the ultrasound-assisted dispersive-magnetic nanocomposites-solid-phase microextraction (UA-DMNSPME) method was performed to extract AO and MB from aqueous samples by applying magnesium oxide nanoparticles (MgO-NPs). The proposed technique is low-cost, facile, fast, and compatible with many existing instrumental methods. Parameters affecting the extraction of AO and MB were optimized using response surface methodology (RSM). Short extraction time, low experimental tests, low consumption of organic solvent, low limits of detection (LOD), and high preconcentration factor (PF) was the advantages of method. The PF was 44.5, and LOD for AO and MB was 0.33 ng mL -1 and 1.66 ng mL -1 , respectively. The linear range of this method for AO and MB were 1-1000 ng mL -1 and 5-2000 ng mL -1 , respectively. In addition, the relative standard deviation (RSD; n = 5) of the mentioned analytes was between 2.9% and 3.1%. The adsorption-desorption studies showed that the efficiency of adsorbent extraction had not declined significantly up to 6 recycling runs, and the adsorbent could be used several times. The interference studies revealed that the presence of different ions did not interfere substantially with the extraction and determination of AO and MB. Therefore, UA-DMNSPME-UV/Vis method can be proposed as an efficient method for preconcentration and extraction of AO and MB from water and wastewater samples., (© 2022. The Author(s).)