The aim of this study is to evaluate the presence of perfluoroalkyl substances (PFASs) in the blood of 46 residents from Barcelona and to study the factors that affect exposure. Compounds analysed included perfluorooctane sulfonate (PFOS), perfluorohexane sulfonate (PFHxS), perfluorobutane sulfonate (PFBS), perfluorooctanoate acid (PFOA) and perfluorononanoate acid (PFNA). Blood was liquid–liquid extracted and PFASs were determined by liquid chromatography coupled to tandem mass spectrometry. Good recoveries (between 97 ± 14 and 105 ± 13 %) were obtained and method detection limits were from 0.03 to 0.07 ng mL−1. ΣPFASs ranged from 0.11 to 4.37 ng mL−1. PFOS was the main compound detected at 0.09–3.35 ng mL−1, followed by PFOA and PFHxS. PFBS and PFNA were seldom detected. Working conditions, smoking and gender did not cause any significant differences among ΣPFASs levels in the blood while age and parity produced decreased concentrations. On the other hand, laboratory working conditions produced significant higher PFOA levels compared to the general population. Compared to other studies, the PFASs levels in blood from Barcelona residents is low (mean ΣPFASs of 1.67 ± 0.88 ng mL−1) and with little variation among the studied population.