1,3-Diphenylisobenzofuran (DPBF) is a fluorescent molecule which is believed to react highly specifically toward reactive oxygen species such as singlet oxygen (1O2), hydroxy (HO·), alkyloxy (RO·), and alkylperoxy (ROO·) radicals. In all cases the reaction product is 1,2-dibenzoylbenzene. In order to prove that DPBF gives the same product in contact with reactive nitrogen species, its reaction with nitrogen dioxide radical has been studied in 2,2,4-trimethylpentane using the steady-state fluorescence method and mass spectrometry. The progress of the studied reaction was measured by observation of changes in fluorescence intensity of DPBF after addition of nitrogen dioxide (NO2). The rate constants of DPBF fluorescence decay affected by NO2 have been determined. Experiments were conducted over the temperature range of 13–37 °C and for NO2 concentrations from 0.02 to 0.14 mmol dm−3. It has been found that the reaction between 1,3-diphenylisobenzofuran and nitrogen dioxide proceeds in two steps. The first step is a very rapid reaction whose rate could not be measured under established experimental conditions. The second step is slower. The reaction product was identified by registration of mass spectra. The probable reaction mechanism is proposed.