A label-free and specific FRET-based interleukin-6 (IL-6) aptasensor was developed using a DNA aptamer modified with nitrogen-doped carbon quantum dots (NCDs) and gold nanoparticles (AuNPs) as a donor-quencher pair. The assayed target was capable of disrupting the donor–acceptor assemblies yielding a concentration-related fluorescence recovery of NCDs ( λ em = 445 nm and λ ex = 350 nm). By designing two different probes, the interaction of DNA aptamers with IL-6 protein was studied using FRET efficiency. It appeared that the sensing probes showed slightly different sensing profiles. One of the aptasensors showed a linear response of 1.5–5.9 pg/mL for IL-6 with a coefficient of determination of R 2 ≥ 0.99 and the a detection limit of 0.82 pg/mL (at S/N = 3). The experimental results indicated that the biosensor can be applied to determine IL-6 in human serum (with recovery of 95.7–102.9%). Due to the high sensitivity, excellent selectivity, and simplicity of the procedure, this strategy represents a promising alternative for IL-6 sensing in clinical applications. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00604-022-05570-5.