A simple and efficient sample extraction and preconcentration method based on reversed-phase ionic liquid dispersive liquid-liquid microextraction (RP-IL-DLLME) had been developed and used to quantify the domoic acid in human urine samples. The analysis was performed by ultra-performance liquid chromatography and photodiode array detection. During the procedure, hydrophilic ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate [C4mim] BF4 as dispersive solvent and NaOH solution was chosen as extraction solvent. Some important parameters in the method were investigated to get high enrichment factors. Under optimal conditions, the linearity of the method was in the range of 0.1–10 ng mL−1 and the correlation coefficient was above 0.9996. The relative standard deviations (RSDs) of the developed methods for intra-day (n = 5) and inter-day (n = 5) precision ranged from 1.9 to 3.9%. Meanwhile, limit of detection (LOD) was 0.03 ng mL−1(S/N = 3) and that of quantification (LOQ) was 0.1 ng mL−1(S/N = 10) with the enrichment factors (EF) being 230. Eventually, the proposed method was successfully applied to the determination of Dominic acid in human urine samples.