In the current study, a robust dispersive solid-phase extraction (dSPE) strategy using reduced graphene oxide-zinc oxide (rGO-ZnO) nanocomposite as the sorbent was proposed for separation, purification and enrichment of 12 mycotoxins in Coptidis rhizoma (Huanglian). The targeted mycotoxins included aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1, alternariol-methylether, mycophenolic acid, ochratoxin A, penitrem A, nivalenol, zearalenone and zearalanone. The rGO-ZnO nanocomposite was successfully synthesized through hydrothermal process by a modified Hummers method, and further characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), FTIR spectroscopy, ultraviolet-visible spectroscopy and X-ray diffraction (XRD). Several key parameters affecting the performance of the dSPE approach were extensively investigated, and after optimization, acetonitrile/water/formic acid (80/19/1, v/v/v) as the extraction solution, 2% acetonitrile as the adsorption solution, 15 mg rGO-ZnO as the sorbent, n-hexane as the washing solution, and methanol/formic acid (99/1, v/v) as the desorption solution presented an excellent purification and enrichment efficiency. Under the optimal dSPE procedure followed by analysis with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), adequate linearity (R ≥ 0.991), high sensitivity (limit of quantification in the range of 0.09-0.41 µg kg), acceptable recovery (70.3-105.7%) and satisfactory precision (RSD 1.4-15.0%) were obtained. The analysis of 12 selected mycotoxins was also carried out in real Coptidis rhizoma (Huanglian) samples for applicability evaluation of the established method.