In this work, strong electronegativity fluorine-doped carbon nanodots (FCDs) with distinctive dual-ultraviolet-emission characteristics was easily synthesized by a simple solvothermal approach using 4-fluorophenethylamine as the only precursor. Experimental results demonstrate that the FCDs exhibits dual-ultraviolet luminous peaks at 286 and 386 nm due to the participation of fluorine and nitrogen atoms, under 265 nm excitation. Next, the FCDs can be further served as an efficacious ratiometric photoluminescence (PL) probe for tryptophan (Trp) analysis based on the energy/electron transfer of the inner filter effect mechanism between FCDs and Trp. Under optimized experimental conditions, a wide linear relationship obtained between the PL386nm/PL286nm ratio of FCDs and the concentrations of Trp in the range of 0–200.0 μM, with the limit of detection of 0.31 µM (3σ/k). Moreover, the developed ratiometric probe was smoothly applied for estimating Trp in serum samples, revealing promising application opportunities in biological fluids. This work further demonstrates the importance of heteroatom species in regulating the physicochemical properties of carbon materials.