In the present research, dynamic electromembrane extraction (DEME) was introduced for the first time for extraction and determination of ionizable species from different biological matrices. The setup proposed for DEME provides an efficient, stable, and reproducible method to increase extraction efficiency. This setup consists of a piece of hollow fiber mounted inside a glass flow cell by means of two plastics connector tubes. In this dynamic system, an organic solvent is impregnated into the pores of hollow fiber as supported liquid membrane (SLM); an aqueous acceptor solution is repeatedly pumped into the lumen of hollow fiber by a syringe pump whereas a peristaltic pump is used to move sample solution around the mounted hollow fiber into the flow cell. Two platinum electrodes connected to a power supply are used during extractions which are located into the lumen of the hollow fiber and glass flow cell, respectively. The method was applied for extraction of amitriptyline (AMI) and nortriptyline (NOR) as model analytes from biological fluids. Effective parameters on DEME of the model analytes were investigated and optimized. Under optimized conditions, the calibration curves were linear in the range of 2.0-100μgL(-1) with coefficient of determination (r(2)) more than 0.9902 for both of the analytes. The relative standard deviations (RSD %) were less than 8.4% based on four replicate measurements. LODs less than 1.0μgL(-1) were obtained for both AMI and NOR. The preconcentration factors higher than 83-fold were obtained for the extraction of AMI and NOR in various biological samples.