The epithelial cell adhesion molecule (EpCAM) is frequently overexpressed in colorectal cancer (CRC) and thus represents an attractive therapeutic target. However, the clinical efficacy of EpCAM-targeting antibodies has been limited by suboptimal binding properties and reliance on immune effector mechanisms. Here, we describe the development and characterization of anti-EpCAM #126, a fully human monoclonal antibody isolated from the OPAL phage-display library, and its corresponding antibody–drug conjugate (ADC). Anti-EpCAM #126 exhibited substantially higher binding affinity than adecatumumab, primarily owing to a markedly lower dissociation rate. High-density peptide microarray analysis and alanine-scanning mutagenesis identified NGSKLGRRAK (residues 74–83) within the EpCAM extracellular domain as the dominant epitope, with S76, R81, and K83 serving as key contact residues. Binding was conformation-dependent and markedly decreased following trypsin-mediated proteolytic cleavage of the epitope region. Anti-EpCAM #126 demonstrated robust staining of CRC xenograft and patient-derived tumor tissues and exhibited low cytokine release in human peripheral blood mononuclear cells and mouse models. Following the confirmation of efficient receptor-mediated internalization, anti-EpCAM #126 was conjugated to monomethyl auristatin E (MMAE) through a protease-cleavable valine–alanine linker, generating an ADC with a controlled average drug-to-antibody ratio of 4.31 while retaining nanomolar EpCAM-binding affinity. The resulting ADC exhibited potent cytotoxicity against EpCAM-expressing CRC cell lines and achieved 84.96% tumor growth inhibition in a patient-derived xenograft model at 10 mg/kg, whereas the unconjugated antibody showed no antitumor activity. These findings suggest that anti-EpCAM #126-MMAE is a high-affinity, epitope-specific ADC with promising preclinical efficacy against EpCAM-positive CRC.