Anammox bacteria tend to self-aggregate, and biofilm-based anammox processes are widely used as sustainable alternative methods for nitrogen removal from wastewater. However, the mechanism for the initial adhesion of anammox bacteria remains unclear. In this work, extracellular polymeric substances (EPS) were extracted separately from anammox granules and then characterized by multi-methods. The adhesion properties of anammox consortia to biotic and abiotic surfaces were examined separately before and after the extraction of three stratified EPS, using aggregation assays and a quartz crystal microbalance technique with dissipation monitoring, respectively. The extraction of each of the three stratified EPS gradually increased the initial aggregation of anammox consortia from 77.6 ± 3.0% to 85.2 ± 2.6%. Fourier transform infrared spectrometry confirmed that the aggregation of anammox consortia depended largely on the interactions between functional groups on the cell surfaces. All three stratified EPS had positive effects on the initial adhesion rate and mass of anammox consortia to abiotic surfaces. More importantly, the structure of the adhered layer was more compact before the extraction of each of the three stratified EPS. We therefore hypothesized that the initial adhesion among anammox consortia was due to the ability of the anammox bacteria to express adhesion molecules on the bacterial surfaces and that the three stratified EPS were excreted to adhere inert particulates and form a compact biofilm structure. This study clarifies the role of stratified EPS on the initial formation of anammox biofilms and provides a theoretical basis for accelerating the formation of anammox biofilms. Display omitted •Role of stratified EPS in anammox consortia adhesion was studied separately.•The aggregation of anammox consortia was attributed to the bacteria themselves.•All stratified EPS stimulated anammox consortia adhering to abiotic surfaces.•All stratified EPS favored formation of a compact biofilm structure.