This study reports an Fe(3+)-functionalized carbon quantum dots (Fe(3+)-functionalized CQDs) for the highly sensitive and selective detection of ascorbic acid (AA) in rat brain microdialysates based on the specific redox reaction between iron(III) ions and AA. The carbon quantum dots (CQDs) were synthesized by one-step pyrolysis of a small organic molecules i.e. tris(hydroxymethyl)aminomethane (Tris). Fe(3+) can tightly chelate to the surface of CQDs by the hydroxyl group to form Fe(3+)-functionalized CQDs while the fluorescence of CQDs can be effectively quenched by Fe(3+) via Fluorescence resonance energy transfer (FRET). The fluorescence of the Fe(3+)-functionalized CQDs can be sensitively turned on by AA to give an "on-off-on" fluorescence response through the oxidation-reduction between Fe(3+) and AA since the produced Fe(2+) has much lower chelating ability to CQDs and the fluorescence of CQDs can be restored. This Fe(3+)-functionalized CQDs based nanoprobe shows high selective and sensitive response in the concentration of AA ranging from 0.1 μM to 50 μM with the detection limit as lower as 9.1 nM, which is lower than other assays. Finally, the proposed fluorescent probe was successfully applied to direct analysis of AA in biological fluids, i.e. rat brain microdialysates, and may pave a new route to the design of effective carbon quantum dots-based fluorescence probes for other bioassay.