In this work, the upconversion Er3+/Yb3+ co-doped ZrO2 nanoparticles were synthesized and characterized. The nanoparticles were obtained via sol-gel method. To achieve upconversion activity ZrO2 nanoparticles were doped with lanthanide ion pair of the Yb3+ and Er3+ and further calcined at 800°C. According to X-ray powder and high-resolution transmission electron microscopy the well-crystallized monoclinic nanoparticles were formed, which size ranges from 20 to 30 nm for undoped ZrO2 and from 30 to 40 nm for doped ZrO2. The calcined Er3+/Yb3+ co-doped ZrO2 nanoparticles demonstrated an upconversion luminescence visible by the bare eye. Namely, when excited with NIR-laser, the nanoparticles emit visible green light. Moreover, the Er3+/Yb3+ co-doped ZrO2 nanoparticles showed red (650–673 nm) and green (520–570 nm) upconversion luminescence due to the 4F9/2 → 4I15/2 of and (2H11/2 and 4S3/2) → 4I15/2 transitions of Er3+ ion, respectively. The obtained nanoparticles exhibited intense luminescence alongside with low toxicity according to colorimetric assay for cellular growth and survival. It was concluded that Er3+/Yb3+ co-doped ZrO2 nanoparticles are perfect prospective bioimaging agents for using as extremely sensitive detectors of biomarkers or excessive contrast deep tissue indicators.