Rare earth upconversion fluorescent nanomaterials have drawn great attention as novel fluorescent probes because of their many unique merits, such as superior photostability, narrow emission spectra, long lifetime and larger Stokes shift. By using near infrared laser as excitation light source, it is feasible for the upconversion fluorescent nanomaterials as excellent fluorescent probes for in vivo fluorescence imaging due to the high light penetration depth in biological tissues, absence of photodamage to live organisms, low autofluorescence background. Recent progress of preparation and biological application of rare earth upconversion fluorescent nanomaterials is reviewed in this paper.