Along with the advancement of DNA nanotechnology, DNA nanomaterials have been extensively explored and applied in the biomedical field. DNA nanoflowers (DNFs), characterized as flower-shaped nanocrystals, have attracted notable interest in the biomedical field because of their large surface area relative to volume and significant surface roughness; enabling high loading capacity. Due to their unique sequence programmability, function designability, and biocompatibility, DNFs have increasingly been researched in biosensing, bioimaging and, drug delivery and therapy. Self-assembly of DNFs enables them to maintain DNA stability and provides additional functions of metal ions, which are difficult to obtain through conventional methods. However, some challenges must be addressed, such as the interaction between DNA and inorganic material and controlling the structural features like petals and DNA sequence. In this review, we discussed the designability of DNFs, we subsequently discuss the synthetic methods and controllable parameters. Then, we enumerate recent applications of DNFs in biosensing, bioimaging, drug delivery and therapy and, templating novel functional materials. Finally, we provide conclusion remarks and perspectives for future directions.