Lanthanide-doped upconversion nanoparticles (UCNPs) can convert two or more lower-energy near-infrared photons to a single photon with higher energy, which makes them particularly suitable for constructing nanoprobes with large imaging depth and minimal interference of autofluorescence and light scattering from biosamples. Furthermore, they feature excellent photostability, sharp and narrow emissions, and large anti-Stokes shift, which confer them the capability of long-period bioimaging and real-time tracking. In recent years, UCNPs-based nanoprobes (UC-nanoprobes) have been attracting increasing interest in biological and medical research. Signal contrast, the ratio of signal intensity after and before the reaction of the probe and target, is the determinant factor of the sensitivity of all reaction-based probes. This progress report presents the methods of constructing UC-nanoprobes, with a focus fixed on recent strategies to improve the signal contrast, which have kept on promoting the bioapplication of this type of probe.