<p> MOF-801@MXene nanocomposites are&#xa0;introduced as a new solid-phase microextraction coating. This structure was easily prepared by one-pot solvothermal route. The MOF-801@MXene, MOF-801, and MXene were characterized by various analysis techniques such as field emission scanning electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, X-ray powder diffraction, and Brunauer–Emmett–Teller. The coating was used in the headspace solid-phase microextraction of methadone and tramadol in biological samples. The separation and determination of the analytes were performed by gas chromatography–mass spectrometry. The effective parameters on the extraction efficiency of the analytes, such as extraction time and temperature, desorption time and temperature, salt concentration, and NaOH concentration, were optimized by experimental design method. Under optimal conditions, low limits of detection in the range 0.03–0.15&#xa0;µg L⁻¹, wide linearity in the range 0.10–250.00&#xa0;µg L⁻¹, and good reproducibility (RSD = 5.3 to 7.4% for <i>n</i> = 3) were achieved. Under optimal conditions, microextraction of methadone and tramadol was performed in real hair, urine, and plasma samples, and satisfactory results were obtained.</p> Graphical Abstract <p></p>