Monodisperse spherical Y2O2S:Yb,Ho nanocrystals with particle size about 50 nm were prepared by a modified homogeneous precipitation method combined with low-temperature sulfurization process. The Y2O2S:Yb,Ho nanocrystals exhibit an intense green emission assigned to the Ho3+ ions 5F4, 5S2 → 4I15/2 transition under 980 nm infrared pump. The upconversion luminescence brightness of Y2O2S:Yb,Ho is 526 Cd/m2 under 4.63 W/cm2 pump density, indicating that the as-prepared Y2O2S:Yb,Ho nanocrystals can meet the requirement of in vivo imaging. The formation mechanism of monodisperse spherical oxysulfide nanoparticles is also discussed.