The single-stage configuration of the partial nitritation (PN)–anaerobic ammonium oxidation (AMX) process is preferred due to low pH fluctuation and the low concentration of inhibitory NO2-. The novel core-shell structure integrates ammonia-oxidizing reaction in the outer layer to protect AMX bacteria in the core gel bead from oxygen inhibition and provide NO2-. Here, the initial activity of ammonia-oxidizing bacteria (AOB) in the outer layer was assessed by the specific oxygen uptake rate (OUR). AOB activity was positively correlated with biomass loading in the outer layer. A maximum OUR of 10.5 mg-DO/L∙h was achieved with AOB loading of 0.23 mg-VSS per bead. During AOB enrichment, the NO2- production rate was linearly increased up to 0.65 kg-N/m3-d for 17 days (PVA hydrogel packing ratio=17%). The maximum oxygen penetration depth (OPD) was reduced from 966.7±52.3 μm before enrichment to 333.3±36.4 μm after the AOB enrichment. The liable bacteria were observed within a range of 165 μm of the outer layer using the live and dead cell staining. Based on the OPD and liable cell distribution results, a minimal outer layer thickness of approximately 350 μm was suggested to facilitate a successful PN–AMX process.