Lanthanide-doped upconversion nanomaterials that convert low energy excitation wavelength to high energy emission have attracted considerable attention due to the advantages of high penetration depth and high resistance to autofluorescence interference capability. This review highlights the literature of the past several years covering recent advances of upconversion nanoprobes for biodetection. We begin by discussions of the key points in fabrication of lanthanide-activated nanoprobes. We further conduct a careful evaluation of various detection applications of upconversion nanoprobes, including temperature, ions, small molecules, DNA, enzyme, virus, and etc. We finish the discussion by assessing current challenges and future prospects of upconversion nanoprobes for fundamental studies and clinical applications.