Lanthanide-doped up-conversion nanoparticles (UCNPs) provide a remote temperature sensing approach to monitoring biological microenvironments. In this research, the UCNPs of NaYF:Yb, Er@NaYF:Yb, Nd with hexagonal (β)-phase were synthesized and applied in cell temperature sensing as well as imaging after surface modification with meso-2, 3-dimercaptosuccinic acid. In the core-shell UCNPs, Yb ions were introduced as energy transfer media between sensitizers of Nd and activators of Er to improve Eremission and prevent their quenching behavior due to multiple energy levels of Nd. Under the excitations of 808 nm and 980 nm lasers, the NaYF:Yb, Er@NaYF:Yb, Nd nanoparticles exhibited an efficient green band with two emission peaks at 525 nm and 545 nm, respectively, which originated from the transitions of H → I and S → I for Er ions. We demonstrate that an occurrence of good logarithmic linearity exists between the intensity ratio of these two emission peaks and the reciprocal of the inside or outside temperature of NIH-3T3 cells. A better thermal stability is proved through temperature-dependent spectra with a heating-cooling cycle. The obtained viability of NIH-3T3 cells is greater than 90% after incubations of about 12 and 24 (h), and they possess a lower cytotoxicity of UCNPs. This work provides a method for monitoring the cell temperature and its living state from multiple dimensions including temperature response, cell images and visual up-conversion fluorescent color.