Ferrous ion (Fe2+) is an important component of hemoglobin and plays a role in transporting O2 to human tissues. If iron deficiency is present, iron deficiency anemia may occur, so it is critical to develop sensitive and accurate methods to detect Fe2+. Herein, a novel luminescence energy transfer (ET) system has been designed for the sensitive detection of Fe2+ by a single-particle enumeration (SPE) method in the near-infrared (NIR) region through combining NIR-to-NIR β-NaGdF4:Yb,Tm@NaYF4 upconversion nanoparticles (UCNPs) and IR-808 dye. IR-808 dye can quench the luminescence of the UCNPs because of the efficient overlap between the absorption spectrum of IR-808 and the emission spectrum of the UCNPs. When Fe2+ and H2O2 are added to the system, the Fenton reaction produces hydroxyl radicals (˙OH). The generated ˙OH reacts with IR-808 and the structure of IR-808 is destroyed. As a result, the ET process is suppressed, causing recovery of the luminescence of the UCNPs, which is reflected as an increase in the number of luminescent particles. Accurate quantification of Fe2+ is achieved by statistically counting the target concentration-dependent luminescent particles. Under the optimal conditions, the linear detection range of Fe2+ is 5-10 000 nM, which is much wider than the ensemble luminescence spectra measurements in bulk solution. Moreover, this strategy can be applied to detection in serum samples with satisfactory results.