The detection of circulating tumor cells (CTCs) is crucial for cancer management but remains challenging due to their rarity and heterogeneity. Alkaline phosphatase (AP) activity, particularly from cancer-associated isoforms ALPP and ALPG, offers a promising functional biomarker alternative. This study developed and validated a highly sensitive assay for detecting AP-expressing circulating tumor-related materials (CTRMs). Magnetic nanoparticles were conjugated with an optimized BG2-PEG-biotin aptamer for specific CTRM capture, with systematic optimization of probe design, blocking reagents, and background suppression. The assay demonstrated excellent linearity and reproducibility (%CV = 5.6%). Clinical validation across lung cancer (LC) and other cancers revealed significantly elevated AP activity in LC (p = 0.0372) and other cancer patients (p = 0.0576) versus healthy donors. Most notably, AP activity was substantially higher in tyrosine kinase inhibitor (TKI)-resistant patients (p = 0.0001). ROC analysis showed exceptional performance for distinguishing LC from healthy donors (AUC = 0.9340) and identifying TKI resistance (AUC = 0.9198). While no significant difference was found between CTC-positive and negative groups, AP activity strongly correlated with quantitative CTC burden in LC (r = 0.485, p = 0.000) and TKI-resistant subgroups (r = 0.369, p = 0.003). Longitudinal monitoring demonstrated that dynamic AP activity changes reflected treatment response. This study establishes AP activity as a sensitive, specific functional biomarker for CTRM detection that effectively predicts TKI resistance and dynamically monitors tumor burden, highlighting its significant clinical potential for non-invasive cancer management.