<p>Neurotransmitters (NTs) are essential for modulating nerve signal transmission in the spinal cord, and this study aims to develop a highly sensitive, rapid, and accurate method for analyzing NTs in rat spinal cord tissue. This advancement is crucial for improving clinical diagnosis and management of neurological disorders. Ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-QqQ/MS²) in conjunction with ultra-ionic liquid dispersive liquid-liquid microextraction (UA-MIL-DLLME) were employed to extract 20 NTs. Among the two magnetic ionic liquids (MILs) tested, [P_6,6,6,14]₂[CoCl₄] was chosen as the extraction solvent due to its distinct properties, including visual recognition, paramagnetism, and high extraction efficiency. The method features efficient magnetic separation using an external magnetic field. Meanwhile, the color of the ionic liquid itself makes the extraction process easier to observe. To enhance extraction efficiency, the hydrophilic ionic liquid [BMIM]BF₄ was utilized as the dispersion solvent, and parameters such as MIL type, solvent amount, extraction time, salt concentration, and pH were systematically optimized. The resulting method demonstrated high precision, a broad linear range, and low detection limits, with satisfactory recovery rates for all 20 NTs analyzed. Given its exceptional analytical performance, this technology has broad prospects in the analysis of the nervous system.</p>