Bifidobacteria are members of the human gut microbiota and have shown to exert beneficial effects on their host. Certain strains have a long history of safe and effective use as probiotics. Due to the lack of efficient genetic tools, however, little is known about the molecular mechanisms on which these health-promoting properties are based, thus limiting the synthetic biology applications in bifidobacteria. Here, we discuss the recent development of genetic tools and their engagement in engineering bifidobacteria for food and biomedical applications, from eliminating antibiotic resistance mobile elements and improving robustness to preventing pathogen infections and delivering therapeutics for cancer treatment. In addition, we highlight the application of emerging genome engineering techniques for manipulating the bifidobacterial genome. Finally, we provide our perspective on the future development of synthetic biology techniques and programmed probiotic bifidobacteria with enhanced robustness and designer functionalities.