The C-terminal coiled-coil region of mouse and human cartilage matrix protein (CMP) self-assembles into a parallel trimeric complex. Here, we report a general strategy for the development of highly stable trimeric targeting ligands (tribodies), against epidermal growth factor receptor (EGFR) and prostate-specific membrane antigen (PSMA) as examples, by fusing a specific target-binding moiety with a trimerization domain derived from CMP. The resulting fusion proteins can efficiently self-assemble into a well-defined parallel homotrimer with high stability. Surface plasmon resonance (SPR) analysis of the trimeric targeting ligands demonstrated significantly enhanced target-binding strength compared with the corresponding monomers. Cellular-binding studies confirmed that the trimeric targeting ligands have superior binding strength toward their respective receptors. Significantly, the EGFR-binding tribody was considerably accumulated in the tumor of mice bearing xenografted EGFR-positive tumors, indicating its effective cancer-targeting feature under in vivo conditions. Our results demonstrate that CMP-based self-assembly of tribodies can be a general strategy for the facile and robust generation of trivalent targeting ligands for a wide variety of in vitro and in vivo applications.