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Carbon dots as new nanomaterials, have been widely used in rapid detection because of their nondestructive, real-time detection characteristics. Improving the sensitivity and selectivity of the method in complex real samples is new challenge and requirement for sensing technology. Here, we report an ultrasensitive fluorescence immunoassay (FIA) for trace diethyl phthalate (DEP) using red carbon dots@SiO2 (R-CDs@SiO2) as tags. SiO2 as a nanocarrier can effectively improve the bio-functionalization and utilization rate of carbon dots. Moreover, several R-CDs embedded in SiO2 nanospheres can magnify the fluorescence signal and improve sensitivity. R-CDs@SiO2 conjugate anti-DEP antibody (Ab) as fluorescent immunosensor, which can specifically recognize DEP. Under optimization conditions, the detection limit (LOD) of this FIA was calculated as 0.0011 ng/mL. In addition, the recoveries of this established FIA ranged from 96.8 to 108.5%, showing satisfactory accuracy. Compared with GC-MS/MS (LOD µg/mL), the sensitivity of the FIA was significantly improved. As a result, the FIA developed using R-CDs@SiO2 as tags has a high potential for determining trace DEP. [ABSTRACT FROM AUTHOR]