As a carrier of genetic information, DNA is a versatile module for fabricating nanostructures and nanodevices. Functional molecules could be integrated into DNA by precise base complementary pairing, greatly expanding the functions of DNA nanomaterials. These functions endow DNA nanomaterials with great potential in the application of biomedical field. In recent years, functional DNA nanomaterials have been rapidly investigated and perfected. There have been reviews that classified DNA nanomaterials from the perspective of functions, while this review primarily focuses on the preparation methods of functional DNA nanomaterials. This review comprehensively introduces the preparation methods of DNA nanomaterials with functions such as molecular recognition, nanozyme catalysis, drug delivery, and biomedical material templates. Then, the latest application progress of functional DNA nanomaterials is systematically reviewed. Finally, current challenges and future prospects for functional DNA nanomaterials are discussed.