A supramolecular solvent microextraction method was used for the fast and selective preconcentration separation of malathion, and then ultra-performance liquid chromatography-tandem mass spectrometry was applied for the detection of malathion. The supramolecular solvent is a suitable medium for malathion collection from the sample extract. The results showed that the quantitative recovery of malathion was obtained at pH 4 with heptanol-tetrahydrofuran as the supramolecular solvent. The preconcentration of malathion by the developed microextraction method was established within 10 min. The limit of detection and the quantification limit were 1.4 and 4.2 mu g/L, respectively. A preconcentration factor of 40 was obtained, and the relative standard deviation was <7%. The developed supramolecular solvent microextraction method was applied for different real samples, including fruits, vegetables, and water samples.