The authors describe a fluorometric aptamer based assay for detecting β-lactoglobulin by using carbon dots (C-dots) as a signal indicator. The aptamer was immoblized on magnetite (FeO) nanoparticles (MNPs), and the C-dots served as a label for the complementary oligonucleotide (cDNA). The assay is based on the hybridization that takes place between aptamer and cDNA. In the presence of β-lactoglobulin (β-LG), the aptamer preferentially binds to β-LG, and this leads to a partial release of the C-dots-cDNA into the solution. After magnetic separation, the supernatant of the solution contains the released C-dots-cDNA which are quantified by fluorometry, best under excitation/emission wavelengths of 354/447 nm. Under the optimal conditions, the fluorescence intensity is proportional to the logarithm of the β-LG concentration in the 0.25 to 50 ng mL range, with a 37 pg mL detection limit. The method was successfully applied to the determination of β-LG in hypoallergenic formulations, and the results demonstrated that this assay is a promising tool in food quality control. Conceivably, it also provides the opportunity for detection of other analytes. Graphical abstract Schematic of a novel aptamer based fluorometric β-lactoglobulin assay based on the use of magnetite (FeO) nanoparticles (MNPs) and carbon dots (C-dots). C-dots were used as a signal indicator and FeO MNPs acted as a magnetic separator. This assay exhibits high sensitivity and selectivity with a detection limit as low as 37 pg mL.