Aptamers and aptamer-drug conjugates (ApDCs) have shown some success as targeted therapies in cancer theranostics. However, their stability in complex media and their capacity to evade lysosomal breakdown still need improvement. To address these challenges, we herein developed a one-step self-assembly strategy to improve the stability of aptamers or ApDCs, while simultaneously enhancing their delivery performance and therapeutic efficiency through a lysosome-independent pathway. This strategy involves the formation of stable complexes between disulfide monomer and aptamers (Sgc8) or ApDCs (Gem-Sgc8). Self-assembled Sgc8 NPs resisted nuclease degradation for up to 24 h, whereas the aptamer alone degraded within just 3 h. These self-assembled Sgc8 NPs, as well as Gem-Sgc8 NPs, demonstrated enhanced binding capabilities compared to Sgc8 aptamers or Gem-Sgc8 alone. Furthermore, lysosome-independent cellular uptake was significantly improved, which in turn increased the therapeutic efficacy of Gem-Sgc8 NPs by 2.5 times compared to Gem-Sgc8 alone. In vivo results demonstrated that Gem-Sgc8 NPs can effectively suppress the growth of tumors. The same self-assembly strategy was successfully applied to other aptamers, such as MJ5C and cMET, showing the generalizability of our method, Overall, this aptamer self-assembly strategy not only overcomes the limitations associated with instability and lysosomal degradation but also demonstrates its broad applicability, highlighting its potential as a promising avenue for advancing targeted cancer theranostics. STATEMENT OF SIGNIFICANCE: We developed a one-step self-assembly strategy to improve the stability of aptamers or ApDCs and enhance their drug therapeutic efficiency through a lysosome-independent pathway. The stability of self-assembled Sgc8 nanoparticles (NPs) was significantly improved. The resulting Sgc8 NPs or GEM-Sgc8 NPs exhibited enhanced binding ability compared to Sgc8 aptamers or GEM-Sgc8 alone, and they also facilitated lysosome-independent cellular uptake, resulting in a 2.5-fold increase in therapeutic efficacy of GEM-Sgc8-NPs. The same self-assembly strategy was successfully applied to other aptamers, such as MJ5C and cMET, showing the generalizability of our method.