<span class="paragraphSection"><div class="boxTitle">Abstract</div>Aptamers are programmable molecular recognition elements with broad utility in diagnostics, therapeutics, and synthetic biology. However, many aptamers suffer from insufficient affinity due to rapid target dissociation, and no general strategy currently exists to overcome this limitation. Here, we report a symmetry-guided assembly approach that enhances aptamer affinity by suppressing the dissociation rate constant (<span style="font-style: italic;">k</span>_off). Three identical aptamer units are spatially organized into a flexible trivalent assembly to enable kinetic cooperativity through rapid rebinding. Applied to aptamers targeting SARS-CoV-2 spike (both trimeric and monomeric S1 subunit), VEGF₁₆₅ (dimeric), and cardiac troponin I (monomeric), the resulting trimers exhibited dissociation constants (<span style="font-style: italic;">K</span>_d) in the low pM range and <span style="font-style: italic;">k</span>_off values in the 10⁻⁶ s⁻¹ range, over 100-fold improvements relative to monomers. In a serum-based VEGF₁₆₅ assay, the trimeric aptamer improved detection sensitivity by 30-fold. This modular, chemistry-based strategy is applicable to existing aptamers and establishes dissociation suppression as a general principle for engineering ultrahigh-affinity aptamers.</span>