Enzymatic modification has emerged as a crucial technique for enhancing the physicochemical attributes of starch. In particular, the short-chain glucans (SCGs) obtained from the debranching of native starch has been utilized to produce functional micro- and nano-structures through their inherent self-assembly property. However, a significant challenge remains in controlling the self-assembly kinetics of SCGs, which often results in undesirable heterogeneous structures. This study explores the factors governing SCG self-assembly and their impact on the formation of monodisperse starch nanoparticles (SNPs). We discovered that incomplete removal of the debranching enzyme during SCG self-assembly leads to particle aggregation, with the degree of aggregation dependent on the concentration of remaining enzyme. Furthermore, the initial SCG concentration significantly influences SNP growth; high concentration results in gel-like structures, while lower concentration produces discrete and spherical nanoparticles. Complete dispersion of growth species, SCGs, before self-assembly through appropriate high-temperature heating was found to be critical for uniform nuclei formation, thereby resulting in the production of well-defined nanoparticles. Systematically manipulating these kinetic factors allowed us to fabricate monodisperse SNPs with an average size of 200 nm. This study advances our understanding of SCG self-assembly kinetics, providing valuable insights for the precision-controlled synthesis of starch-based nanoparticles.