Lithium regulates the concentration of nitric oxide in the human body and a high dose of nitric oxide causes multiple sclerosis (MS). Also, the amount of manganese in the cerebrospinal fluid alters the metabolic reactions associated with MS. In this study, the mixture of the ammonium pyrrolidine dithiocarbonate (APDC), the hydrophobic ionic liquid [HMIM][PF6] and acetone coated on the surface of graphene oxide nanoparticles (GONPs) and used for separation Li and Mn in human samples by ultrasound assisted-dispersive-ionic liquid-micro-solid phase extraction technique (USA-DIL-μ-SPE) at pH 6.0. After extraction and back-extraction, the amount of lithium and manganese in the blood, serum and urine samples was determined by the flame and the graphite furnace atomic absorption spectroscopy (F-AAS, GF-AAS), respectively. By optimizing parameters, the LOD, Linear ranges (LR) and preconcentration factor (PF) for Li and Mn ions were obtained (0.03 mg L-1, 0.25 μg L-1), (0.1-0.4 mg L-1, 0.08-1.5 μg L-1) and 10, respectively (%RSD<5). The capacity adsorption of APDC/IL/GONPs and GONPs was achieved (148.5 mg g-1, 122.3 mg g-1) and (41.3 mg g-1, 33.7 mg g-1) for Li and Mn ions in a static system, respectively. This method was successfully validated by spiking samples and certified reference materials (CRM).