Natural product evodiamine derivatives exhibit multitarget bioactivities as dual TOPO I/II inhibitors, demonstrating remarkable potential in antitumor applications. Based on the evodiamine derivative D7-03, six derivatives were synthesized. In vitro screening showed that D7-09 had the strongest anti-tumor activity (IC50 = 9.75-26.11 nM) but poor hydrophobicity and solubility. D7-03, with nM-level cytotoxicity, better physicochemical properties, and high yield, was chosen as the core payload for ADC construction. The active molecule D7-03 was further explored as an antibody-drug conjugate (ADC) payload by constructing four linker-toxin complexes. These complexes were conjugated with trastuzumab to generate ADC candidate molecules. Notably, Ab-DL07-D7-03 exhibited superior activity (IC50 = 8.369 nM and 4.899 nM in HCC1954 and NCI-N87 cells, respectively) compared to the control group Ab-LC08-SN38. This study is the first application of evodiamine derivatives as TOPO I / II dual inhibitors in the ADC field, which not only provides a new strategy for the development of ADC payloads but also enriches the innovative application of natural product small molecules in tumor-targeted therapy.