Room temperature ionic liquids (RTILs) are considered "Green Solvents" for their thermally stable and non-volatile properties. They are employed to replace traditional volatile organic solvents in solvent extraction; organic synthesis, and electrochemistry. In the present work, a simple, selective, sensitive and economical microextraction methodology was developed based on the separation and pr-concentration of RTILs for trace cadmium (Cd) and copper (Cu) determination in biological samples. Cd and Cu pre-concentration was established using the RTIL(1-butyl-3-methylimidazolium hexafluorophosphate ([C4mim] [PF6]) with dithizorie complex. For the quantitative recovery of Cd and Cu, the effects of the parameters inducing the extraction efficiency and its subsequent determinations, i.e., pH, amount of complexing reagent, volurne of RTILs and dispersant solvent, sample Volume,, extraction time, and temperature Were studied. For Cd and Cu determination under the optimized conditions, the detection limits (LOD) of 0.05 and 0.08 mu g/L and quantification limits (LOQ) of 0.15 and 0.25 mu g/L, respectively, were obtained. An enrichment factor of 75 was achieved for both analytes and the relative standard deviation was < 3%. BCR 144R sewage sludge of domestic origin certified reference material (CRM) was used for validation of the present method. The proposed RTIL. microextraction method was applied for the pre-concentration of Cd and Cu in biological samples with satisfactory results.