Streptavidin and its homologs (together referred to as streptavidin) are widely used in molecular science owing to their highly selective and stable interaction with biotin. Other factors also contribute to the popularity of the streptavidin-biotin system, including the stability of the protein and various chemical and enzymatic biotinylation methods available for use with different experimental designs. The technology has enjoyed a renaissance of a sort in recent years, as new streptavidin variants are engineered to complement native proteins and novel methods of introducing selective biotinylation are developed for in vitro and in vivo applications. There have been notable developments in the areas of catalysis, cell biology, and proteomics in addition to continued applications in the more established areas of detection, labeling and drug delivery. This review summarizes recent advances in streptavidin engineering and new applications based on the streptavidin-biotin interaction.