Dimeric prodrug self-assembly nanoparticles (DPS NPs) present promising avenues for chemotherapeutic delivery, yet optimizing the linker design for effective SN38 delivery remains challenging. We developed various SN38 dimeric prodrugs with differing linker lengths to explore how linker length impacts DPS NP performance. Our study reveals that linker length critically affects the nanoparticles assembly stability and activation efficiency. Specifically, too short linkers compromise assembly stability and lead to premature drug activation in the bloodstream, raising safety concerns. Conversely, too long linkers hinder both assembly stability and activation efficiency within tumor cells, diminishing anti-tumor effectiveness. The optimal linker (C12) achieved the best balance, ensuring robust assembly stability and high activation efficiency, thereby enhancing anti-tumor efficacy while maintaining a favorable safety profile. This work underscores the significance of linker length in designing effective DPS NPs for cancer treatment.