Background: In recent years, non-invasive imaging technologies for early cancer detection have drawn worldwide attention. In this study, an antinucleolin aptamer, AS1411, was successfully conjugated to BODIPY-labeled chitosan and studied on T47D and HEK-293 cell lines. Methods: After conjugation of the aptamer to chitosan nanoparticles and purification, its structure was confirmed using atomic force microscopy (AFM), electrophoretic light scattering (ELS) and dynamic light scattering (DLS). Results of AFM, DLS and ELS of both conjugation and chitosan were compared for confirmation of conjugation. Conjugates were mixed with BODIPY FL fluorescent dye, purified and lyophilized. The labeled conjugate was characterized using Fourier-transform infrared spectroscopy, ultraviolet-visible spectroscopy, ELS and DLS. In vitro cellular uptake and cytotoxic effects of BODIPY-labeled chitosan-AS1411 aptamer conjugates were evaluated using the XTT assay on T47D and HEK-293 cells and flow cytometry on T47D cells. Results: The data showed that uptake of BODIPY-labeled chitosan-AS1411 aptamer conjugate was satisfactory. Moreover, there was no statistically significant cytotoxicity of the conjugate on either cell line. Conclusion: The outcomes confirmed the potential application of this new targeted imaging agent as a novel cancer diagnostic agent for molecular imaging.