Aptamers, despite their specific targeting capabilities and widespread applications in various research domains, face a significant hurdle in the biomedical research area due to their rapid degradation by nucleases. To address this challenge, this study introduces an innovative development in the form of polymeric aptamer probes (PAPs) designed to enhance in vivo cancer tissue recognition and targeting. This study outlines the synthesis of PAPs, which leverage the strain-promoted alkyne-azide cycloaddition (SPAAC) strategy to construct these nanoprobes. By sequentially linking individual DBCO or N3 group-decorated AS1411 aptamers that target nucleolin overexpressed on tumor cells, the resulting PAPs exhibit significantly enhanced stability against enzymatic degradation and superior binding affinity and internalization ability compared to single aptamers across a range of cancer cell lines. In vivo experiments have further validated the superior tumor targeting and retention capabilities of the prepared PAPs, thus underscoring their potential for precise cancer diagnosis and therapy.