Engineering inverse 3D polymeric microfilms with controlled spatial hierarchy is both highly challenging and critically important at the intersection of biology and materials science. These structures hold significant potential for enhancing selective cell–surface interactions, including cell adhesion and growth. Protein-modified inverse 3D polymeric microfilms can further promote selective cell capture and adhesion. In this study, we report the fabrication of inverse 3D polymeric microfilms using composite polymeric–bioligand conjugated films designed to enhance the capture of circulating tumor cells (CTCs) from the blood of cancer patients. The microfilms were developed using functionalized poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA), mediated with the ligand transferrin. Protein immobilization on the films was achieved by conjugating transferrin (Tf), collagen (Co), and bovine serum albumin (BSA) to promote cellular adhesion and capture. The films were characterized using scanning electron microscopy (SEM), attenuated total reflectance infrared spectroscopy (ATR-IR), and contact angle measurements, revealing micropores ranging from 18–26 μm. Confocal laser scanning microscopy (CLSM) demonstrated enhanced cell attachment on the polymeric-blend microfilms, confirming improved cell adhesion, capture, and the ability of cells to proliferate within the 3D structure. The inverse 3D polymeric microfilms achieved an 80% cell capture efficiency with cultured cancer cells. In clinical utility, their CTC capturing efficiency was comparable to OncoDiscover® CTC enumeration technology. These inverse 3D polymeric microfilms represent a novel ‘lab-on-a-chip’ platform capable of enabling CTC enumeration for monitoring minimal residual disease (MRD), tracking metastatic progression, evaluating treatment response, and enabling early detection of relapse.