Targeted delivery or "smart delivery" of pharmaceutical or imaging agents and even entire cells such as stem cells is an emerging trend in modern biotechnology. A binding ligand such as an antibody that can specifically bind to receptors expressed at a disease site is an essential component of such constructs. Different chemical methods have been widely used to apply antibodies for delivery systems; however, they typically result in impairment or loss of antibody functionality. Enzyme-mediated conjugation approaches have been developed to overcome this major disadvantage of conventional chemical methods. Sortase, an enzyme derived from Staphylococcus aureus, is able to provide a biochemically robust, highly reproducible, and site-specific coupling method for the conjugation of antibodies to pharmaceutical agents, nanoparticles, and cells for drug delivery, molecular imaging, and cell homing. Here, we review the use of sortase and other enzyme-based methods as bioconjugation tools with a focus on cardiovascular applications.