Fusion protein linkers are essential components in the design of stable, bioactive recombinant fusion proteins. This review discusses the properties, design, and functions of linkers derived from naturally-occurring multi-domain proteins and empirical linkers used in recombinant fusion proteins. Linkers can be classified into flexible, rigid, and in vivo cleavable types. Flexible linkers, composed of small or polar amino acids like Gly and Ser, provide flexibility and solubility, while rigid linkers, often α-helical or Pro-rich, offer structural stability and distance between domains. In vivo cleavable linkers allow for the release of functional domains under specific conditions, such as proteolytic cleavage, to enhance bioactivity and target specific sites in vivo. Linkers improve the folding, stability, and expression of fusion proteins by maintaining proper distances between domains, reducing interference, and facilitating correct folding. They also enhance bioactivity by enabling the in vivo release of functional domains. For example, the use of helical linkers in G-CSF-Tf fusion proteins significantly improved bioactivity, while Pro-rich linkers in HSA-IFN-α2b fusion proteins enhanced antiviral activity. Linkers can also target fusion proteins to specific sites in vivo by incorporating protease-sensitive sequences that are cleaved under pathological conditions, such as in cancer or inflammation. This targeted delivery increases the efficacy of therapeutic proteins by ensuring they are active at the site of action. The design of linkers is guided by their structural properties, including length, hydrophobicity, and secondary structure. Databases and computational tools aid in selecting optimal linkers for specific applications. Overall, linkers play a crucial role in the development of effective recombinant fusion proteins, enhancing their stability, bioactivity, and targeted delivery.Fusion protein linkers are essential components in the design of stable, bioactive recombinant fusion proteins. This review discusses the properties, design, and functions of linkers derived from naturally-occurring multi-domain proteins and empirical linkers used in recombinant fusion proteins. Linkers can be classified into flexible, rigid, and in vivo cleavable types. Flexible linkers, composed of small or polar amino acids like Gly and Ser, provide flexibility and solubility, while rigid linkers, often α-helical or Pro-rich, offer structural stability and distance between domains. In vivo cleavable linkers allow for the release of functional domains under specific conditions, such as proteolytic cleavage, to enhance bioactivity and target specific sites in vivo. Linkers improve the folding, stability, and expression of fusion proteins by maintaining proper distances between domains, reducing interference, and facilitating correct folding. They also enhance bioactivity by enabling the in vivo release of functional domains. For example, the use of helical linkers in G-CSF-Tf fusion proteins significantly improved bioactivity, while Pro-rich linkers in HSA-IFN-α2b fusion proteins enhanced antiviral activity. Linkers can also target fusion proteins to specific sites in vivo by incorporating protease-sensitive sequences that are cleaved under pathological conditions, such as in cancer or inflammation. This targeted delivery increases the efficacy of therapeutic proteins by ensuring they are active at the site of action. The design of linkers is guided by their structural properties, including length, hydrophobicity, and secondary structure. Databases and computational tools aid in selecting optimal linkers for specific applications. Overall, linkers play a crucial role in the development of effective recombinant fusion proteins, enhancing their stability, bioactivity, and targeted delivery.