Abstract: Bentonite colloids act as potential carriers for the migration of radioactive nuclides, and their aggregation behavior directly influences the long-term storage of high-level radioactive waste(HLW) geological disposal repositories. Here, nanoscale effects of alkaline earth metal cations on the aggregation behavior of single-layer bentonite colloids(1 g/L, pH=7.5) were investigated by synchrotron small-angle X-ray scattering(SAXS). The fine structure of colloidal aggregates was analyzed in relation to reaction times, ion concentrations, and ion types using SAXS data analysis methods, including Kratky plot analysis, Lorentzian peak fitting, and estimation of the average stacking layer number(N). The single-layer bentonite colloids, prepared via centrifugation followed by dialysis, exhibit a lamellar thickness of 1.07 nm. When the Ca²⁺ concentration exceeds 0.44 mmol/L, it induces the bentonite colloids to form a periodic face-to-face stacked structure through “ion bridging”. Both prolonged reaction time and increased Ca²⁺ concentration lead to a reduction in the d-space and an increase in N. As the reaction time extends from 10 minutes to 3 days, the d-space contracts from (1.883±0.004) nm to (1.853±0.002) nm, while N increases from (7.2±0.1) to (8.7±0.1) layers, reaching equilibrium after 2-3 days. When the Ca²⁺ concentration rises from 0.48 mmol/L to 6.00 mmol/L, the d-space decreases from (1.888±0.006) nm to (1.847±0.001) nm, and N grows from (4.2±0.1) to (9.7±0.1) layers. In contrast, Na⁺ induces significantly weaker aggregation; even at high concentration(50 mmol/L), it only promotes the formation of a loose “card-house” structure by edge-face interactions, attributed to the limited compression of the colloidal double layer. Four alkaline earth ions(Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺) induce face-to-face stacking. Mg²⁺-bentonite colloids exhibit the largest d-space((1.904±0.002) nm) and the smallest N((8.5±0.1) layers), whereas Ba²⁺-bentonite colloids show the smallest d-spacing((1.848±0.002) nm) and the largest N((9.8±0.1) layers). The d-space positively correlates with the hydrated ionic radius of the alkaline earth metal ions. The aggregation capability of \mathrmM^2+_\mathrmhydrated is Ba²⁺＞Sr²⁺＞Ca²⁺＞Mg²⁺, as smaller hydrated ions with higher charge density and greater polarizability exhibit stronger aggregation effects. Groundwater in the Beishan area contains abundant alkaline earth cations(primarily Ca²⁺ and Mg²⁺), which destabilize suspended bentonite colloids in fracture water and reduce their potential to facilitate radionuclides migration. Synchrotron SAXS offers unique advantages for probing the dynamic structural evolution of colloids in solution environments, owing to its high intensity, superior spatiotemporal resolution, and in situ detection capability. The methodology established and the conclusions drawn in this work provide new research insights and data support for the safety assessment of HLW geological repositories.