Ionic liquid-based surfactants (ILBSs) represent a new class of hybrid amphiphilic systems that integrate the physicochemical properties of ionic liquids (ILs)—such as high ionic conductivity, negligible vapor pressure, thermal stability, and structural tunability—with the self-assembly behavior of classical surfactants. By rational molecular design of cationic (imidazolium, pyridinium, ammonium, phosphonium), anionic, or zwitterionic headgroups coupled with hydrophobic chains or functional tails, ILBS can be engineered to display tailored interfacial activity, aggregation behavior, and biological interactions. Their ability to form micelles, vesicles, liquid-crystalline phases, and nanoarchitectures enables efficient encapsulation, solubilization, and controlled release of hydrophobic and hydrophilic biomolecules. Recent advances demonstrate that ILBS can offer enhanced biocompatibility, intrinsic antimicrobial and membrane-active properties, improved DNA/RNA complexation efficiency, and enhanced enzyme stabilization under stress environments. These multifunctional molecular assemblies are increasingly explored for applications in drug and gene delivery, gene transfection, protein refolding, tissue engineering, biosensing, biocatalysis, and bio-separation systems. Moreover, tunable physicochemical parameters such as alkyl chain length, headgroup charge density, hydrogen-bonding capability, and counter-ion identity play critical roles in governing cytotoxicity, bio-membrane interactions, biodegradability, and functional performance. Despite promising biological activity and self-assembly versatility, key challenges remain in controlling long-term toxicity, ensuring biodegradability, improving colloidal stability in physiological media, and achieving scalable, cost-efficient synthesis. Structure–property–bioactivity relationships, guided by computational modeling, molecular dynamics simulations, and high-throughput design strategies, are expected to accelerate the development of next-generation ILBS. Future research efforts will focus on designing stimulus-responsive, task-specific ILBS with precision biocompatibility, minimal environmental impact, and tailored functionalities to advance their translation into biomedicine and biotechnology.