Anaerobic　ammonium　oxidation　(anammox)　is　an　energy　saving　and　environmentally　friendly　technique　for　wastewater　treatment.　Sludge　adsorption　is　an　important　process　after　organics　enter　the　anammox　reactor.　The　extracellular　polymeric　substances　(EPS)　of　anammox　sludge　play　a　key　role　on　the　activity　of　anammox.　This　study　utilized　Fourier-transform　ion　cyclotron　resonance　mass　spectrometry　(FT-ICR-MS)　to　investigate　molecular-level　compositional　characteristics　of　the　stratified　EPS　of　the　anammox　sludge,　and　to　explore　the　adsorption　preference　of　each　EPS　layer　to　refractory　dissolved　organic　matter　(DOM)　during　anammox　treatment.　Results　showed　that　the　adsorbed　component　by　the　tightly　bound　EPS　layer　was　dominantly　composed　of　lipids　and　proteins,　with　nearly　80%　of　formulas　being　CHO　and　CHON.　The　outer　layers　(slime,　loosely　bound-EPS)　preferentially　adsorbed　the　compounds　with　comparatively　higher　aromatic　and　unsaturated　degrees,　with　S-containing　formulas　and　lignin　being　the　predominant　components.　The　newly　produced　formulas　in　the　effluent　were　inferred　to　be　associated　with　the　anammox　treatment　performance.　The　refractory　DOM,　with　smaller　molecular　weight　and　high　reductive　degree,　seemed　to　experience　a　single　adsorption　by　the　stratified　EPS,　and　became　part　of　the　anammox　sludge.　©　2020　Elsevier　Ltd