This study presents a comparative kinetic assessment of four representative harmful algal bloom (HAB) isolates from the Yangtze River Estuary using controlled monoculture experiments with defined nitrogen (N) substrates. For each species, we measured Michaelis-Menten apparent kinetic parameters (Vₘₐₓ, Kₛ) employing ¹⁵N tracer techniques, and logistic growth parameters (K, r) under standardized laboratory conditions across three N sources, specifically nitrate (NO₃⁻), urea, and dissolved free amino acids (DFAA). Key findings reveal marked species-substrate differentiation. Skeletonema costatum exhibited the highest half-saturation constant for NO₃⁻ among all treatments, while Chaetoceros curvisetus achieved measurable growth and uptake on organic-N, with urea and DFAA Vₘₐₓ values of 32.5 and 34.6 × 10⁻³ h⁻¹ respectively, substantially exceeding those for NO₃⁻. Prorocentrum shikokuense showed relatively uniform Vₘₐₓ across substrates but exceptional NO₃⁻ affinity, whereas Karenia mikimotoi exhibited negligible NO₃⁻ uptake but extreme DFAA affinity. Notably, all four species demonstrated relatively strong DFAA efficiency. When positioned within published HAB kinetic compilations, the isolates occupied divergent positions. C. curvisetus urea-Vₘₐₓ exceeded the 75^th percentile of global diatom values, while P. shikokuense NO₃⁻-Kₛ fell below the 5^th percentile. Generalized additive modeling of field N uptake rates identified environmental covariation, specifically temperature (~22°C), salinity (25–26 thresholds), and substrate colimitation. These quantitative physiological benchmarks provide testable boundary conditions for predictive modeling of HAB dynamics under changing N stoichiometry in eutrophic estuaries.