Double-emulsion droplets have found widespread applications in various engineering and biomedical fields because of their capability in encapsulating different components in each layer. The conventional double-emulsion method is the two-stage stirring emulsification method, which suffers from poor monodispersity and low encapsulation efficiency. With recent advances in microfabrication, some novel methods for fabricating double-emulsion droplets have been developed, including microfluidic emulsification (double-T-junction microchannel, double-cross-shaped microchannel and several three-dimensional microchannels), membrane emulsification and coaxial electrospraying. These methods have shown significantly improved droplet features (e.g., size, size uniformity, thickness of each layer, generation throughput capability). Herein, this paper first reviews the state-of-art approaches for fabricating double-emulsion droplets and discusses their advantages and disadvantages. The applications of double-emulsion droplets in biomedical fields, including cell encapsulation, drug delivery and controlled release, and synthetic biology are also discussed. In conclusion, future perspectives are given.