<p>Partial nitrification (PN) as one of the most efficient and energy-saving processes, has received much attention for process application. However, ensuring its stability remained a significant challenge. This study investigated the effects of reduced nitrogen loading rate (NLR: 0.328–0.057 kg N/(m³·d)) on substrate transformation linked with excitation-emission matrix (EEM) property, nitrification kinetic and functional bacteria. PN was rapidly initiated under higher NLR condition within 23 cycles with nitrite (NO₂⁻-N) accumulation ratio (NAR) of 73.35% in the continuous-flow, and the inflection points of DO, pH, and ORP accurately marked the process of ammonium (NH₄⁺-N) oxidation and NO₂⁻-N generation in the typical cycle. EEM fluorescence analysis indicated that the main components of soluble microbial products (SMPs) and extracellular polymeric substances (EPS) transformed form tyrosine-like protein, tryptophan—like protein and fulvic acid-like organics to humic acid-like organics, and the fluorescence intensity of SMPs increased significantly under declined NLR. Nitrification kinetic parameters proved that AOB showed higher substrate affinity (<i>K</i>_N,AOB &lt; <i>K</i>_N,NOB, e.g., 1.65 vs. 17.79 mg/L) and maximum specific oxidation rate (<i>q</i>_max,AOB &gt; <i>q</i>_max,NOB, e.g., 15.15 vs. 11.44 mg N/(gVSS·h)) for efficient PN, revealing the reasons why AOB achieved the dominant position in Phase III. Moreover, <i>Nitrosomonas</i>, which was mainly responsible for NO₂⁻-N accumulation, sufficiently enriched (1.12% → 16.94% → 8.25%) and thereby promoted effective retention of functional bacteria. Meanwhile, <i>Nitrospira</i> was eluted (0.56% → 0.31% → 0.49%) and contributed to the stable maintenance of NAR at lower NLR levels. The practical applications and challenges of the PN coupling processes were further summarized for low-strength wastewater.</p>