Vertically‐ordered mesoporous silica films (VMSF, also named as silica isoporous membranes) have shown tremendous potential in the field of electroanalytical sensors due to their unique features in terms of controllable and ultrasmall nanopores, high molecular selectivity and permeability, and mechanical stability. This review will present the recent progress on the biomedical analytical applications of VMSF, focusing on the small biomolecules, diseases‐related biomarkers, drugs and cancer cells. Finally, conclusions with recent developments and future perspective of VMSF in the relevant fields will be envisioned.