The mainstream partial nitritation/anammox (PN/A) process has been intensively studied but its stability remains a key challenge. It is shown here that biofilm thickness can exhibit a critical role in controlling the process stability of mainstream PN/A against dissolved oxygen (DO) variation. In a laboratory moving bed biofilm reactor (MBBR), PN/A performance was initially established in 200 days by controlling a low DO of 0.13 ± 0.07 mg O2/L in the bulk liquid, which deteriorated with an increase of DO (0.35 ± 0.13 mg O2/L) for over two months, and then rapidly recovered in a month with the initial low DO level re-applied. Biofilm thickness of PN/A carriers was measured during the experiment, which became significantly thinner (367 ± 146 μm) at mainstream conditions. The thin thickness primarily decreased the in-situ consumption rate of nitrite, rather than ammonium, when DO increased from 0.1 to 0.4 mg O2/L, due to that the thin thickness can only restrict anammox capacity. These results illustrated the role of biofilm thickness in regulating PN/A performance and microbial activities. Further investigation using an established model revealed the joint contribution of biofilm thickness and DO concentration to PN/A process, while particularly, the biofilm thickness can determine the optimal DO level for maximizing the nitrogen removal efficiency and system robustness against DO variation. These results highlight the need of considering biofilm thickness in PN/A process optimization and stability improvement in low-strength wastewater treatment. Display omitted •N-removal efficiency >80% is achieved via PN/A in a mainstream MBBR.•The mainstream PN/A is stably realized at DO concentration of 0.13 ± 0.07 mgO2/L.•Biofilm thickness controls the maximal capacity of anammox rather than AOB or NOB.•A narrow DO range (0.15-0.20 mgO2/L) is required for high N-removal in thin biofilm.