The competency of polyelectrolyte multilayer films (PEM) self-assembly to modulate the surface architecture at the molecular level has emerged as a captivating technique for various biomedical applications. PEM advancement in bioapplications covered the development of bio-sensors, stimuliresponsive drug-delivery systems, surface modification for creating antibacterial surfaces, and tissue-engineering architectures. Different PEM blends, from natural to synthetic ones, have been explored to make a free-standing version of the PEM stack. This review provides a deeper understanding of the free-standing PEM (FS-PEM), their fabrication strategies, and essential properties for specified bio-applications. The most crucial aspect of fabricating FS-PEM is choosing an application-specific fabrication protocol to delaminate the PEM stack from the underlying substrates. Post-fabrication, there are important considerations to improve the mechanical stability of these FS-PEMs by crosslinking or adding desired nanoparticles. Overall, essential insights are discussed here for using FS-PEM for specific bio-application, which has received little attention in the material science community until now.