Alizarin dye is toxic and has a negative influence on human life and the environment. Consequently, the scientific community faces a difficult issue in developing efficient approaches for removing alizarin from water streams. Six distinct deep eutectic solvents (DESs) containing different hydrogen bond acceptors (HBAs), namely trioctylphosphine, trioctylamine and trihexylamine, and two hydrogen bond donors (HBDs), namely salicylic acid and malonic acid, were used to rapidly remove alizarin from high concentration solutions up to 2000 mg/L at room temperature using the liquid–liquid micro-extraction method (LLE). DES-3 had the highest extraction efficiency for alizarin among the other synthesized DESs. The effect of process variables such pH, contact time, dye initial concentration, volume ratio, temperature and salt on alizarin extraction efficiency from water stream was explored, optimized and reported. Statistical analysis was conducted to ensure the accuracy of values for the optimized parameters. For a 1000 mg/L solution of alizarin with a DES/alizarin volume ratio of 1:10 at room temperature, the maximum elimination of 98.02 percent was achieved in 5 min. FTIR was used to analyze the structural properties of DES and the interaction between DES and alizarin. The thermal stability of DES-3 was determined using thermogravimetric analysis (TGA) and indicated that DES-3 has excellent thermal stability up to 320 °C. Human saline was used to test the toxicity of the synthesized DES in vitro. It was determined that synthesized DES is less harmful and more effective at removing alizarin.