Stable inhibition of nitrite-oxidizing bacteria (NOB) is a significant challenge in achieving partial nitrification (PN) and partial nitrification-anaerobic ammonia oxidation (PNA). Growing evidence suggested that NOB can develop resistance to suppression over time, leading to the re-enrichment of NOB within reactors. To address these issues, this study aimed to achieve stable PN by regulating SRT to selectively washout NOB during the lag phase of activity recovery following FA/FNA exposure. The effects of this new strategy on nitrifying bacteria, sludge characteristics, and microbial interspecies relationships were investigated over a 150-day trial. Under the new strategy operation, the effluent ammonia, nitrite, and nitrate concentrations were 4.72 mg/L, 51.81 mg/L, and 3.30 mg/L, respectively, at a dissolved oxygen (DO) concentration of 0.18 mg/L, with a nitrite accumulation rate (NAR) remaining above 95%. This was attributed to the increasing relative abundance of ammonia-oxidizing bacteria (AOB) (Nitrosomonas) up to 25.86% and the decreasing relative abundance of NOB (Nitrospira, Nitrobacter and Candidatus Nitrotoga) to below the detection limit (1%) during the stabilization period. Analysis of the PN sludge characteristics revealed a tendency for sludge to form loosely structured aggregates, facilitating the potential rapid start-up of the PNA biofilm system or granular sludge. These findings suggest that the new strategy is a straightforward and effective method for achieving PN.