Abstract: A single-domain antibody (sdAb) is the variable region extracted from antibodies with only heavy chains found in camelid serum. Single-domain antibodies have the characteristics of easy expression, good solubility, great stability and weak immunogenicity. Since single-domain antibodies have higher penetration in tissues than conventional antibodies and can be cleared by the kidneys quickly, they are ideally suited for various biotechnologies and clinical treatment of tumors. The monovalent sdAb could be coupled to radioisotopes, fluorescent dyes, biotin, magnetic beads, affinity matrices and so on. A complex molecular-conjugated sdAb can be used for in vivo multiple modes tumor imaging. In the basic research, development of probe and other fields, single-domain antibody has a broad prospect in application. This review summarizes the molecular features and the applications in tumor molecular imaging of single-domain antibodies.