Constructing nitrogen-doped porous carbons with high specific surface area, rapid mass transfer channels, and positive charge is a crucial requirement for high-performance adsorbents. Herein, by the kinetic self-assembly synthesis strategy, we prepared nitrogen-doped hierarchical porous carbon spheres (N-HPCS) with adjustable pore structure, high specific surface area, and high nitrogen doping content (8.88 %). By using ethylenediamine as an assisted polymerization and assembly agent, the hydrolysis and condensation rate of tetraethyl orthosilicate (TEOS) as the silica source and the condensation rate of 3-aminophenol and formaldehyde as the phenolic resin precursor were controlled by adjusting ammonia volume as the alkaline catalyst to tune kinetic self-assembly of silica and phenolic resin components, thus achieving their simultaneous or sequential nucleus and growth. After carbonization and selective silica etching, three types of carbon nanospheres with center-radial pores, hollow center-radial pores and hollow structure were obtained. High nitrogen doping content endowed the nanospheres with positive charge. Through adsorption experiments on the bovine serum albumin (BSA) and Hemoglobin (Hb) as typical biological macromolecules, hollow carbon nanospheres with center-radial pores exhibited excellent adsorption performance for BSA(622.34 mg g−1) and Hb(759.96 mg g−1). Our fabricated N-HPCS may become a potential candidate for high-performance adsorption materials.