Streptavidin (SA), and other related proteins, has been isolated from a wide range of organisms, including bacteria, fungi, frogs, fish, and birds. Although their original function is not well understood, they have found an important place in biotechnology based on their unique ability to bind biotin molecules with high affinity and specificity. The SA-biotin interaction is robust and easy to incorporate into different designs, and as such, it is used when reliable molecule interaction is needed under poorly controlled experimental conditions. There are continued efforts to engineer these proteins to modulate their size, valency, and affinity, since the optimum molecular properties vary depending on individual applications. This review will describe recent developments in streptavidin engineering to meet these requirements, including those that form novel oligomeric states, e.g., a monomer, have fewer functional biotin-binding sites, or bind biotin with reduced affinity. We also examine various reported applications of both natural or engineered SA constructs in cell biology, biochemistry, genetics, synthetic chemistry, cancer therapy, drug delivery, and nanotechnology to illustrate the breadth of modern science that is advanced by the endogenous and engineered SA-biotin interactions.