Severe side effects of chemotherapy agents on vital organs are the major causes of cancer-related mortality, not merely cancer disease. Encapsulating chemotherapeutic molecules in nanocarriers is a justifiable solution in decreasing the risk of their side effects and boosting the efficiency of treatment. The present study has developed the doxorubicin (DOX)-loaded AS1411 (anti-nucleolin) aptamer surface-functionalized exosome (DOX-Apt-Exo) to treat colorectal cancer in both in-vitro and in-vivo experimental models. HEK293-derived exosomes were loaded with DOX through the incubation method with a nearly 13% encapsulation efficiency. Afterwards, the 5-terminal carboxyl group of AS1411-aptamer was converted into amine-reactive NHS esters with EDC/NHS amide coupling chemistry before being conjugated to the amine groups on the exosome surface. DLS and TEM estimated the designed formulation (DOX-Apt-Exo) size of about 200 nm. Aptamer-binding affinity and cellular uptake of DOX-Apt-Exo by nucleolin-overexpressing cancer cells were depicted through fluorescence microscopy. Comparing the in-vitro cytotoxicity impact of DOX-loaded exosomes, either targeted or non-targeted by MTT assay, clearly verified a high effectiveness of ligand-receptor mediated target therapy. Subsequently, in-vivo experiments which were conducted on four groups of ectopic mouse models of colon cancer (5 in each group) demonstrated the tumor growth suppression through professional long-term accumulation and retention of DOX-Apt-Exo at the tumor site by ligand-receptor interaction. The results suggested that AS1411 aptamer-functionalized exosomes can be recommended as a safe and effective system to site-specific drug delivery in possible clinical applications of colon cancer.