A superparamagnetic iron oxide nanoparticles (SPIONs)/doxorubicin (Dox) co-loaded poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles targeted with AS1411 aptamer (Apt) against murine C26 colon carcinoma cells is successfully developed via a modified multiple emulsion solvent evaporation method for theranostic purposes. The mean size of SPIO/Dox-NPs (NPs) was 130 nm with a narrow particle size distribution and Dox loading of 3.0%. The SPIO loading of 16.0% and acceptable magnetic properties are obtained and analyzed using thermogravimetric and vibration simple magnetometer analysis, respectively. The best release profile from NPs was observed in PBS at pH 7.4, in which very low burst release was observed. Nucleolin is a targeting ligand to facilitate anti-tumor delivery of AS1411-targeted NPs. The Apt conjugation to NPs (Apt-NPs) enhanced cellular uptake of Dox in C26 cancer cells. Apt-NPs enhance the cytotoxicity effect of Dox followed by a significantly higher tumor inhibition and prolonged animal survival in mice bearing C26 colon carcinoma xenografts. Furthermore, Apt-NPs enhance the contrast of magnetic resonance images in tumor site. Altogether, these Apt-NPs could be considered as a powerful tumor-targeted delivery system for their potential as dual therapeutic and diagnostic applications in cancers.