A multifunctional assay with up-/down-conversion (UC/DC) nanohybrids which enables the detection and real-time tracking of hazardous molecules has been developed for use in the field of photoluminescence (PL) point-of-care testing due to its cost and convenience. In particular, innovative approaches such as dual or multimodal imaging and detection under only a single-photon pulse system are highly difficult owing to the issues of device simplification and miniaturization. In this work, we first demonstrated single-photon-driven UC/DC dual-modal PL nanohybrids and showed their high performance in in vivo mercury detection and real-time tracking in a mussel simultaneously. Specifically, UC/DC nanohybrids capable of being stimulated by a single photon were presented via a facile and versatile strategy by combining DC fluorophores for heavy metal ion screening with triplet–triplet annihilation upconversion (TTA-UC) nanocapsules for real-time tracking. By adopting the advantages of the structural transformation of DC fluorophores and highly stable TTA-UC nanocapsules, the outstanding monitoring performance of a standard heavy metal ion (i.e. Hg²⁺) was achieved by a dual-modal PL assay with nanohybrids, exhibiting ultra-sensitivity (under 1 nM) and high-selectivity. Interestingly, their application in the real world was also remarkable in screening and tracking of mercury in mussels. This single-photon-driven UC/DC convergence system will provide powerful analytical methodologies for target detection and real-time tracking in vivo and will attract widespread attention from researchers in the fields of PL nanomaterials and fluorophores.