Novel polyepinephrine-modified NaYF:Yb,Tm upconversion luminescent nanoparticles (UCNP@PEP) were prepared via the self-polymerization of epinephrine on the surfaces of the UCNPs for selective sensing of Fe inside a cell and for intracellular imaging. The proposed UCNP@PEP probe is a strong blue light emitter (λ = 474 nm) upon exposure to an excitation wavelength of 980 nm. The probe was used for detecting Fe owing to the complexation reaction between UCNP@PEP and Fe, resulting in reduced upconversion luminescence (UCL) intensity. The proposed probe has a detection limit of 0.2 μM and a good linear range of 1-10 μM for sensing Fe ions. Moreover, the UCNP@PEP probe displays high cell viability (90%) and is feasible for intracellular imaging. The ability of the probe to sense Fe in a human serum sample was tested and shows promising output for diagnostic purposes. The prepared UCNP@PEP probe was characterized by using UV-visible (UV-Vis) absorption spectrometry, fluorescence (FL) spectrometry, field emission scanning electron microscopy (FE-SEM), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FT-IR).