An ion selective microelectrode (ISME) was fabricated to measure concentrations of ammonium (${NH_4}^+$-N) and nitrate (${NO_3}^-$-N) according to the depth of nitrifying biofilm. The limits of detectability and validity of results were investigated to evaluate the ISME. The electromotive force (EMF) was directly proportional to the ion concentration, and average detection limits of ${NH_4}^+$ and ${NO_3}^-$ ISME were $10^{-5.14}$ and $10^{-5.18}$ M, respectively. The concentrations of ${NH_4}^+ $-N and ${NO_3}^-$-N in various depths on the nitrifying biofilm were measured by the ISME. When a modified Ludzack-Ettinger (MLE) process was operated at an HRT of 6 h, concentration gradients of ${NH_4}^+$-N in the bulk solution and biofilm at depths of $100;{mu}m$ decreased by $70;{mu}M$, while ${NO_3}^-$-N increased by $101;{mu}M$ and remained constant thereafter. At an HRT of 4 h, concentration gradients of ${NH_4}^+$-N at depths of $500;{mu}m$ decreased by $160;{mu}M$ and ${NO_3}^-$-N increased by $162;{mu}M$ and remained constant thereafter. As HRT decreased, the concentration gradients of ${NH_4}^+$-N and ${NO_3}^-$-N between bulk solution and biofilm was higher due to the increase of nitrogen load. Also, the concentration gradients of the ${NH_4}^+$-N and ${NO_3}^-$-N of biofilm in the second aerobic tank were lower than those of the first aerobic tank, suggesting differences of nitrogen load and concentrations of DO between the first and second aerobic tank.