A reliable and sensitive analytical approach has been optimized for the extraction of seven polychlorinated biphenyls (PCBs) from human breast milk. Hollow fiber liquid phase microextraction (HF-LPME) was applied for the first time for the extraction and pre-concentration of the analytes. Analytes were separated by gas chromatography with electron capture detector (GC-μECD) for the sensitive detection and mass spectrometry for the unequivocal identification. A rotable central composite design (RCCD) was performed for the multivariate optimization of the method. The best results were obtained at 40 °C during 30 min and 600 rpm of stirring speed using a hollow fiber length of 5 cm and toluene as an extractant phase and salt addition was not required. The detection limits were in the range 7-14 ng L for PCBs. The coefficients of determination of the calibration curves indicated good linearity (R> 0.96) and the enrichment factors ranged from 74 to 143. This type of study is of great importance due to the deleterious effect that the presence of contaminants can produce in infants health related to the immature character of the defense system. Moreover, exclusive breastfeeding is recommended by neonatologists up to six months of life and as complementary food during the first two years.