Food contaminants pose a danger to human health, but rapid, sensitive and reliable food safety detection methods can offer a solution to this problem. In this study, an optical fiber ratiometric fluorescence sensing system based on carbon dots (CDs) and o-phenylenediamine (OPD) was constructed. The ratiometric fluorescence response of Cu2+ and thiram was carried out by the fluorescence resonance energy transfer (FRET) between CDs and 2,3−diaminophenazine (ox-OPD, oxidized state o-phenylenediamine). The oxidation of OPD by Cu2+ resulted in the formation of ox-OPD, which quenched the fluorescence of CDs and exhibited a new emission peak at 573 nm. The formation of a [dithiocarbamate-Cu2+] (DTC-Cu2+) complex by reacting thiram with Cu2+, inhibits the OPD oxidation reaction triggered by Cu2+, thus turning off the fluorescence signal of OPD-Cu2+. The as-established detection system presented excellent sensitivity and selectivity for the detection of Cu2+ and thiram in the ranges of 1 ∼ 100 μM and 5 ∼ 50 μM, respectively. The lowest detection limits were 0.392 μM for Cu2+ and 0.522 μM for thiram. Furthermore, actual sample analysis indicated that the sensor had the potential for Cu2+ and thiram assays in real sample analysis.