In the present work, pulsed electromembrane extraction (PEME) was performed for the first time, as a new concept of electrically enhanced microextraction method, for extraction and quantification of histidine, phenylalanine and tryptophan in different matrices. PEME offers an alternative to conventional electromembrane extraction (EME), which faces problems such as serious instabilities in the analysis of real samples with high concentration levels of ions. In these samples, increasing of the ion transportation across the liquid membrane results in Joule heating during the extraction process which may follow by punctuation of the organic membrane, increasing of the current level and bubble formation due to electrolysis reactions. A mixture of 2-nitrophenyl octyl ether (NPOE), di-(2-ethylhexyl) phosphate (DEHP) and tris-(2-ethylhexyl) phosphate (TEHP) was immobilized in the pores of hollow fiber as the organic liquid membrane. Other effective parameters such as extraction time, ion balance and pulse frequency were optimized using the experimental design. Extraction recoveries in the range of 7.1-21.6% and satisfactory repeatability (2.1<CV%<4.5) were obtained. Limits of detection were 5, 10 and 30 ng mL(-1) for tryptophan, phenylalanine and histidine, respectively. The method offers acceptable linearity with correlation coefficients higher than 0.9979. Furthermore, the figures of merit of PEME are compared with the results from conventional electromembrane extraction (EME), which proves the advantages of the proposed technique. The method was applied to the determination and quantification of amino acids in foods and biological samples. Also, two-way PEME was employed as a novel approach for highly selective extraction of tryptophan as a model analyte to introduce an interesting ability of the proposed technique.