Biosensors convert target recognition events into measurable signals. Traditional nucleic acid detection methods (e.g., PCR) can be time-consuming and instrument-intensive, highlighting the need for simpler, rapid signal output strategies. In this study, we investigated G-quadruplex DNA structures as a versatile signal output platform in DNA biosensors. Split G-quadruplex constructs (with the 12 guanines divided between two DNA fragments and stabilized by a complementary linker) were tested under various ionic conditions. Signal output was measured either colorimetrically via a G-quadruplex–hemin DNAzyme that catalyzes tetramethylbenzidine (TMB) oxidation (producing a visible color change), or fluorometrically via G-quadruplex–enhanced thioflavin T (ThT) fluorescence. As a result of systematic optimization, we found that an uneven 9:3 split G-quadruplex with a fully complementary linker yielded the highest colorimetric response in a Ca²⁺-rich buffer with 5 μM hemin and a 20-minute reaction time. For fluorescence output, the strongest signals were achieved with a 9:3 split in a Na⁺ buffer and a 6:6 split in a Mg²⁺ buffer, both using 5 μM ThT. These findings demonstrate that tailoring the G-quadruplex structure and reaction conditions can significantly enhance biosensor signal output. The optimized split G-quadruplex system provides a convenient and flexible signal output method, enabling rapid visual detection by color change without specialized equipment as well as sensitive fluorescence detection with instrumentation, thereby potentially simplifying and improving DNA biosensor design for diverse applications.