The unique merits of aptamers, including specificity, high binding affinity, easy cell internalization, and rapid tissue accumulation abilities, have led aptamer-drug conjugates to evolve into one of the most attractive strategies for targeted drug delivery purpose. Nevertheless, the critical role of linkers in regulating of anticancer efficacy of these conjugates, especially those engineered by automated modular synthesis techniques, have been rarely explored. In this work, we utilized Sgc8c aptamer and combretastatin A4 to develop three conjugates with either a phosphodiester bond linker, a disulfide bond linker, or a carbamate linker to study their payload release mechanisms, and the influence on anticancer efficacy. These investigations allowed us to identify the unique activation pathway of the phosphodiester bond linker that is activated both by nucleophilic attack of glutathione (GSH) and degradation caused by phosphodiesterase, which is highly associated with the higher cytotoxicity of conjugate. Importantly, the understanding of the chemistry of phosphodiester bond linker activation allowed us to further design another XQ-2d-CA4 conjugate that can induce pancreatic cancer cells apoptosis in a more efficient manner.