The main challenge for partial nitrification is to reach stable nitrite accumulation, which strongly depends on the nitrite-oxidizing bacteria (NOB) growth in the reactor. The on-line estimation of active biomass may enhance the decision-making process to maintain a high nitrite accumulation in the reactor. In this work, we propose an active biomass estimator based on ASM1 and on-line oxygen uptake rate measurements (OUR-E) in a sequencing batch reactor. In order to validate the OUR-E, two operating scenarios were applied during 200 days of operation: unfavorable (sludge retention time (SRT) = 40 d, pH = 7.6, dissolved oxygen (DO) = 2 mg/L) and favorable for partial nitrification (SRT = 10 d, pH = 8.5, DO = 2 mg/L). Furthermore, a second estimation method based on off-line measurements of N-species concentrations (Nsp-E) was implemented to evaluate the performance of the OUR-E. The OUR-E was able to predict a reduction in the NOB active fraction from 10.3% to 1.6% with nitrite accumulation over 80% when we shifted the operating scenario. Although both estimators predicted similar results, the OUR-E showed a better prediction quality than the Nsp-E, according to Theil's coefficient of inequality.