Chirality exists in every corner of nature and is closely associated with the phenomenon of life. The unique optical properties and enhanced enantioselectivity of chiral nanomaterials mean that they excel in many applications in nanobiology and nanomedicine. Here, we comprehensively review the progress that has been made toward the use of chiral nanomaterials in biosensing, bioimaging, and disease therapies. The construction strategies for three different types of chiral nanomaterials and their chirality-generating mechanisms are first generalized. We then focus on the biological applications of these chiral nanomaterials, ranging from chirality-based ultrasensitive detection and multifunctional imaging to therapeutic purposes. Finally, we present an in-depth discussion of the current challenges and future prospects in the utilization of these exciting materials.