International environmental regulations have created the need for sensitive, accurate, and reproducible analytical tools to measure ultralow sulfur concentrations in fuels, such as diesel and gasoline. In this work, a sensitive, accurate, and reproducible analytical methodology to measure ultralow sulfur concentrations in desulfurized diesels (<0.01 ppb) is presented. The spectroscopic technique used here overcomes the lack of selectivity or signal overlapping that is present in other techniques used nowadays. The methodology consists first of an enzymatic partial oxidation of sulfur compounds of diesel, followed by the selective determination of the sulfur content from the emission spectra of the oxidized diesel using simple regression analysis. As a result of the enzymatic oxidation, we report here that the oxidized diesel exhibits different spectroscopic behaviors, where the oxidized organic sulfur compounds present a characteristic emission band at higher wavelengths. This fact is taken in this study for a selective quantification of sulfur from diesel. Our study is focused on 4,6-dimethyldibenzothiophene (DMDBT) as a model and standard compound for predicting the sulfur content in desulfurized diesel. The correlation coefficient (R-2) between predicted and real sulfur contents was 0.95.