Water-soluble lanthanide (Ln(3+)) doped Bi2O3 nanospheres have been successfully prepared through a solid-state-chemistry thermal decomposition process. The nanospheres exhibit intense upconversion luminescence (UCL) by doping the Ln(3+) (Ln = Yb, Er/Ho/Tm) ions into the Bi2O3 host matrix under 980 nm excitation. The ratio of red/green emission of Bi2O3:Yb(3+)/Er(3+) nanospheres exhibits a significant change as the calcination temperature increases and the value could reach 105.6. Moreover, the UCL of Bi2O3:Yb(3+)/Tm(3+) nanospheres are temperature-sensitive, where the intensity ratios of 799 and 808 nm emissions increase monotonously with temperature. The MTT assay reveals that Bi2O3:Yb(3+)/Tm(3+) nanospheres exhibit good biocompatibility by grafting citric acid molecules on the surface. The application possibility of Bi2O3:Yb(3+)/Tm(3+) nanospheres as bioprobes for optical imaging in vivo is also confirmed by the high-contrast photoluminescence images between the background and the UCL imaging area.