Fluorescence imaging techniques represent essential tools in in vitro, preclinical, and clinical studies. In this study, an improved one-step hydrothermal method to synthesize citric acid (CA) modified α-NaYbF4:2%Er3+nanocrystals was proposed. The introduction of various doping ions into NaYbF4:2%Er3+and the different valence states of the same ions affect both the crystal size and upconversion luminescence. Therefore, we investigated the upconversion luminescence enhancement of NaYbF4:2%Er3+by ion doping and find that the upconversion luminescence intensity of the upconversion nanoparticles (UCNPs) co-doped with 5 mol% Fe2+ions shows the greatest enhancement, especially for red emission at 654 nm. Furthermore, HeLa cells incubated with UCNPs allow for imaging with strong red upconversion emission detection. Confocal laser scanning microscope (CLSM) fluorescent images of HeLa cells indicate that NaYbF4:2%Er/5%Fe2+leads to a clear outline and improves visualization of the cell morphology. In addition, the CA coated NaYbF4:2%Er3+/5%Fe2+nanoparticles and NaYbF4:2%Er3+/5%Fe2+show low cytotoxicity in HeLa cells. Organ imaging reveals the efficiency of these UCNPs to analyze the lungs, liver, and spleen. Together, these results indicate that the Cit-NaYbF4:2%Er3+/5%Fe2+UCNPs are efficient nanoprobes for fluorescence molecular tomography.