Cancer stem cells play critical roles in cancer progression, cancer invasion and metastasis, and cancer recurrence. CD44 is known as a specific surface marker of cancer stem cells, which has been well-studied in cancer invasion and metastasis. Herein, we successfully selected the DNA aptamers for recognizing CD44⁺ cells using Cell-SELEX strategy, in which the engineered CD44 overexpression cells were used as target cells for selection. The optimized aptamer candidate C24S showed high binding affinity with the K_d value of 14.54 nM and good specificity. Then, the aptamer C24S was employed to prepare the functional aptamer-magnetic nanoparticles (C24S-MNPs) for CD44⁺ CTC capture. To investigate the capture efficiency and sensitivity of C24S-MNPs, series of cell capture tests were performed using artificial samples with 10–200 of HeLa cells spiked into 1 mL PBS or PBMCs isolated from 1 mL peripheral blood, obtaining an efficiency of 95% and 90%, respectively. More importantly, we finally explored the facility of C24S-MNPs for CTC detection in blood samples from clinical cancer patients, indicating a potential and feasible strategy for cancer diagnostic technology in clinical applications.