In this work, a single particle imaging method based on the total internal reflection (TIR) imaging platform for the sensing and cell imaging of nitrite (NO₂⁻) in the near-infrared region using cyanine dye-assembled composite upconversion nanoparticles (UCNPs) was developed. The NaYF₄:Yb,Tm@NaGdF₄ UCNPs were synthesized as energy donors, and the cyanine dye IR-798 was prepared as an energy acceptor. Since the absorption spectrum of the cyanine dye IR-798 and the luminescence spectrum of upconversion nanoparticles overlapped effectively, IR-798 quenched the luminescence of the UCNPs. When NO₂⁻ was added to the cyanine dye-assembled composite upconversion nanoparticle system, NO₂⁻ destroyed the conjugate structure of IR-798, so that the luminous intensity of UCNPs could be restored. Based on the mechanism, a quantitative image analysis with high sensitivity, low sample usage, and fast response time using the TIR single particle imaging platform was developed to determine the content of nitrite in human serum samples. In addition, the UCNPs-IR-798 probe was applied to image the exogenous NO₂⁻ content in HeLa living cells based on the single particle imaging platform.