Accurate determination of selenium (Se) species in biological samples is a critical issue because Se commonly occurs at low levels and in diverse species. The method for the full quantification of Se species in serum samples was proposed through combined ion-pair reverse-phase (RP) chromatography and affinity chromatography (AF) hyphenated to inductively coupled plasma-(quadrupole) mass spectrometry (ICP-qMS) with post-column isotope dilution analysis (IDA) and a collision cell technique (CCT). Different Se species like inorganic Se (Se4+ and Se6+), selenocystine (SeCys), selenomethionine (SeMet), selenoprotein P (SelP), selenoalbumin (SeAlb) and glutathione peroxidase (GPx) can be separated and quantified. The proposed methodology was used to qualitatively and quantitatively study the dynamic distribution of Se species in human serum samples from the Hg-contaminated area after supplementation with 100 μg of Se daily as Se-enriched yeast for 180 days. SelP takes up almost half and even more of the total Se and increases with the Se administration. The repeatability in terms of relative standard deviation (R.S.D. %, n = 10) is 6% for GPx and SelP and 5% for SeAlb. The detection limits are 0.1 μg Se L−1 for GPx and other non-retained Se compounds, 1.0 μg Se L−1 for SelP and 1.2 μg Se L−1 for SeAlb, 1.3 μg Se L−1 for inorganic Se; 1.2 μg Se L−1 for SeCys; 1.1 μg Se L−1 for SeMet, respectively.