In this study, a sequencing batch reactor (SBR) was operated to investigate the inhibitory kinetics of free ammonia (FA) on ammonia-oxidizing bacteria (AOB). At the beginning of the experiment, FA concentrations in influent were altered to achieve stable short-cut nitrification and enrich AOB. Nitritation sludge was then employed to study variations in the specific nitrite production rate (SNiPR) during the ammonia oxidation process of batch tests. Furthermore, a kinetic model of FA inhibition on AOB activity was fitted for statistical analysis. Results showed that SNiPR increased rapidly with increase in FA concentration (0.7 mg·L-1 ≤ FA ≤ 50.2 mg·L-1) but decreased with an increase in FA concentration (FA ≥ 50.2 mg·L-1). SNiPR was maintained at 0 g·(g·d)-1 when FA concentration was higher than 687.1 mg·L-1, implying that AOB activity was completely inhibited. Statistical analysis showed that, compared to Haldane, Edwards-1#, Edwards-2#, and Luong inhibition kinetics models, the Aiba model was the most suitable for describing the inhibitory effect of FA on AOB activity. The statistical constants, i.e., residual square sum (RSS) correlation coefficient (R2), F value of the fitting equation, and confidence degree (P) were 0.005, 0.932, 181.7, and 1.06×10-9, respectively. The dynamic constant values, i.e., maximum specific nitrite production rate (rmax), half saturation constant (KS), and inhibition constant (KI) were 0.37 g·(g·d)-1, 11.78 mg·L-1, and 153.74 mg·L-1, respectively.