Nicotinamide adenine dinucleotide (NAD+) and adenosine triphosphate (ATP) are crucial small molecules linked to diverse physiological processes and diseases, making their robust quantification of paramount importance. Herein, we propose a novel label-free fluorescence sensing platform for the quantitative detection of NAD+ and ATP, by seamlessly coupling an intramolecular split G-quadruplex (Intra-SG)-based DNA probe with enzymatic DNA reactions, including high-fidelity cofactor recognition by DNA ligases and the structure-dependent cleavage by Exonuclease III (Exo III). Exo III effectively digests the nicked duplex to release a trigger strand (TS), which subsequently hybridizes with the spacer of the Intra-SG, and triggers the complexation between the Intra-SG and fluorescent dyes (Thioflavin T, ThT) and subsequent fluorescent enhancement. Target-induced ligation yields a repaired duplex that resists Exo III cleavage, resulting in fluorescence quenching. This strategy exhibited linear ranges of 25-300 nM for NAD+ and 50-300 nM for ATP, with detection limits of 12.35 nM and 29.46 nM, respectively. Furthermore, it guarantees exquisite selectivity against structural analogues, and practical applicability in complex biological matrices. Overall, the effectiveness of the Intra-SG-based single probe is validated, and it is anticipated to be integrated with diverse DNA reactions for multifarious bioanalysis.