The fabrication of lanthanide upconversion nanocomposites as probes has become a new research hotspot due to its special advantages via utilizing upconversion luminescence (UCL) as a detection signal. Herein, a hybrid organic dye modified upconversion nanophosphor is successfully developed as a nanoprobe for cysteine/homocysteine. Yolk–shell structured upconversion nanoparticles (YSUCNP) with lanthanide upconversion nanophosphor as moveable core and silica as mesoporous shell are synthesized, and a colorimetric chemodosimeter for cysteine/homocysteine is accommodated in the hollow cavities. Thus, cysteine/homocysteine can be quantitatively detected on the basis of luminescent resonance energy transfer (LRET) in a UCL turn-off pattern. The dye-loaded YSUCNP possess good dispersibility in aqueous solution; thus detection of the targeted molecule can be achieved in pure water. Cellular experiments carried out with laser-scanning upconversion luminescence microscopy further demonstrate that the dye-loaded YSUCNP can serve as an intracellular nanoprobe to detect cysteine/homocysteine. Moreover, this dye-loading protocol can be developed as a common approach to construct other chemodosimeter-modified UCNP hybrid nanoprobes, as proved by a UCL turn-on style sensor for cyanide.Keywords: chemodosimeter; cysteine; homocysteine; LRET; upconverting luminescence