Lactoferrin (Lf) occurs predominantly within milk, coexisting with measurable levels across different glandular products and body fluids. Lf exhibits variation in relative molecular mass, influenced by its biological source and glycosylation profile; nevertheless, it is a close to 80 kDa glycoprotein. Provided that its bioactive structure is preserved, Lf performs a spectrum of physiological roles, comprising antioxidant, antifungal, antiviral, antiapoptotic, and antimicrobial actions. To sustain its bioactivity after isolation and ensure its effectiveness in subsequent applications, optimal conditions must be established throughout the optimization protocol, since inadequate optimization of parameters such as pH, temperature, ion balance, and protease activity may lead to aggregation, denaturation, and deterioration in functional regions, including the iron-binding domains. This review offers a comprehensive framework that associates isolation methodologies with structural integrity, preservation of iron-binding domains, and antimicrobial performance. Ion-exchange, affinity-based, and membrane-based approaches are systematically evaluated from analytical and functional perspectives, thereby yielding a synthesis that facilitates procedure selection and optimization for Lf isolation. In addition, the objectives of analytical characterization techniques implemented following isolation and the broadening scope of biotechnological applications of Lf are outlined.