Liquid biopsy capable of noninvasive and real-time molecular profiling is considered as a breakthrough technology, endowing an opportunity for precise diagnosis of individual patients. Extracellular vesicles (EVs) and circulating tumor cells (CTCs) consisting of substantial disease-related molecular information play an important role in liquid biopsy. Therefore, it is critically significant to exploit high-performance recognition ligands for efficient isolation and analysis of EVs and CTCs from complex body fluids. Aptamers exhibit extraordinary merits of high specificity and affinity, which are considered as superior recognition ligands for liquid biopsy. In this review, we first summarize recent advanced strategies for the evolution of high-performance aptamers and the construction of various aptamer-based recognition elements. Subsequently, we mainly discuss the isolation and analysis of EVs and CTCs based on the aptamer functioned biomaterials/biointerface. Ultimately, we envision major challenges and future direction of aptamer-based liquid biopsy for clinical utilities.