Nucleic acid aptamers hold great promise for therapeutic applications due to their favorable intrinsic properties, as well as high-throughput experimental selection techniques. Despite the utility of the systematic evolution of ligands by the exponential enrichment (SELEX) method for aptamer determination, complementary in silico aptamer design is highly sought after to facilitate virtual screening and increased understanding of important nucleic acid-protein interactions. Here, with a combined experimental and theoretical approach, we have developed two optimal epithelial cellular adhesion molecule (EpCAM) aptamers. Our structure-based in silico method first predicts their binding modes and then optimizes them for EpCAM with molecular dynamics simulations, docking, and free energy calculations. Our isothermal titration calorimetry experiments further confirm that the EpCAM aptamers indeed exhibit enhanced affinity over a previously patented nanomolar aptamer, EP23. Moreover, our study suggests that EP23 and the de novo designed aptamers primarily bind to EpCAM dimers (and not monomers, as hypothesized in previous published works), suggesting a paradigm for developing EpCAM-targeted therapies.