Detection of Fe3+ ion is essential for human health because it is an important element of hemoglobin, which carries oxygen to cells in the body. Here, a 3,3',5,5'-tetramethylbenzidine (TMB) functionalized NaYF4: Yb3+, Er3+@NaYF4 composite upconversion probe was developed, and demonstrated Fe3+ sensing ability with high sensitivity and selectivity. The red emission of upconversion nanoparticles (UCNPs) has a higher penetration depth in tissue than green light and works within the biological window. The obtained hydrophobic NaYF4: Yb3+, Er3+@NaYF4 nanoparticles were treated with HCl to achieve hydrophilic ligand-free nanoparticles with non-saturated metal ions on their surface. Then, a Fe3+ responsive TMB-UNCPs composite luminescence probe was formed through linking TMB onto the ligand-free UCNPs by a coordination bond between the NH2 groups in TMB and the metal ions on the UCNPs. Due to the efficient fluorescence resonance energy transfer from UCNPs to Fe3+-TMB, the obtained probe shows high sensitivity for detecting Fe3+ in the range of 0-100 μM with a detection limit of 0.217 μM. And the color change of the detection system can also be easily recognized by the naked eye. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) experiments and the bioimaging experiments show promising prospects in tissue imaging.