A simple and applied method based on ultrasound assisted-Ionic liquid trap-micro solid phase extraction (USA-ILT-μSPE) was demonstrated for preconcentration/separation of lead (Pb) in human blood and urine samples. By proposed method, the amine-functionalized bimodal mesoporous silica nanoparticles (NH2-UVM7) as solid phase sorbent was used for lead analysis in human biological samples at pH≈7.0. After shaking/centrifuging of sample, the Pb (II) was extracted with NH2-UVM7 without any chelating agents, trapped in ionic liquid phase and settled down in conical centrifuge tube as a residual solution [Pb→:NH2-UVM7]. Finally, the lead ions was back extracted from residual solution and determined by atom trap flame atomic absorption spectrometry (AT-FAAS). Under the optimal conditions, the linear range (LR), limit of detection (LOD) and preconcentration factor (PF) were obtained (10–950μgL−1; 2.0–210μgL−1), (2.8μgL−1; 0.6μgL−1) and (19.8; 99.7) for human blood and urine samples, respectively (% RSD<1, intra-day). The reusability and adsorption capacity of the sorbent were also obtained 24 and 213.8mgg−1, respectively. Validation of methodology was confirmed by certified reference materials (NIST, CRM).