In recent years, two-photon excited (TPE) materials have attracted great attentions because of their excellent advantages over conventional one-photon excited (OPE) materials, such as deep tissue penetration, three-dimensional spatial selectivity and low phototoxicity. Also, they have been widely applied in lots of field, such as biosensing, imaging, photo-catalysis, photoelectric conversion, and therapy. In this article, we review recent advances in vibrant topic of two-photon fluorescent nanomaterials, including organic molecules, quantum dots (QDs), carbon dots (CDs) and metal nanoclus-ters (MNCs). The optical properties, synthetic methods and important applications of TPE nanomaterials in biomedical field, such as biosensing, imaging and therapy are introduced. Also, the probable challenges and perspectives in the forthcoming development of two-photon fluorescent nanomaterials are addressed.