Pancreatic cancer is a highly lethal malignancy associated with tissues of the pancreas. Early diagnosis and effective treatment are crucial to improving the survival rate of patients with pancreatic cancer. In a previous study, we employed the cell-SELEX strategy to obtain an ssDNA aptamer termed XQ-2d with high binding affinity for pancreatic cancer. Here, we first identify CD71 as the XQ-2d-binding target. We found that knockdown of CD71 abolished the binding of XQ-2d and that the binding affinity of XQ-2d is associated with membrane-bound CD71, rather than total CD71 levels. Competitive analysis revealed that XQ-2d shares the same binding site on CD71 with transferrin (Tf), but not anti-CD71 antibody. We then used a surface energy transfer (SET) nanoruler to measure the distance between the binding sites of XQ-2d and anti-CD71 antibody, and it was about 15 nm. Furthermore, we did molecular dynamics simulation to clarify that the spatial structure of XQ-2d and Tf competitively binding to CD71. We also engineered XQ-2d-mediated targeted therapy for pancreatic cancer, using an XQ-2d-based complex for loading doxorubicin (Dox). Because CD71 is overexpressed not only in pancreatic cancer but also in a variety of tumors, our work provides a systematic novel way of studying a potential biomarker and also promising tools for cancer diagnosis and therapy, opening new doors for effective cancer theranostics.