This review article provides an inclusive overview of the intricate interactions amid ionic liquids (ILs) and essential biomacromolecules, mainly hemoglobin (Hb), bovine serum albumin (BSA), human serum albumin (HSA), and calf thymus-DNA (CT-DNA). ILs have recently become a topic of great attention because of their inimitable physicochemical properties and potential uses in different fields. The review systematically explores the binding mechanisms, thermodynamics, and structural changes induced by ILs on Hb, BSA, HSA, and CT-DNA using spectroscopic, thermodynamic, and computational techniques. The article highlighted various experimental and computational methodologies to explore the interactions between ILs and biomacromolecules. It offers an in-depth analysis of the techniques employed to decode the intricate nature of these molecular associations and the influence of ILs along with their structural characteristics on the conformational stability, activity, and functionality of biomacromolecules. This foundational understanding is essential for advancing research and developing strategies that exploit the distinctive properties of ILs to foster innovative and sustainable applications within the biomedical field.