Headspace-solid phase microextraction (HS-SPME) with a polypyrrole (PPy)-coated fiber was applied as a sample preparation method for determination of selenite [Se(IV)] following derivatization with 1,2-diaminobenzene to convert into the piaselenol form and analysis by ion mobility spectrometry (IMS). The HS-SPME-IMS method presents good repeatability (RSDs <6%), simplicity, good sensitivity and short analysis times. The influence of the various analytical parameters on microextraction procedure, i.e. ligand concentration, pH, ionic strength, equilibrium time and temperature has been evaluated and optimized. The calibration graph was linear in the range of 20-320 ng mL(-1) with a detection limit of 12 ng mL(-1). The method was applied for determination of selenium in human serum and environmental surface water samples with satisfactory recovery.