Aim: To develop a new detection technique for ATP in cancer cells using fluorescent biosensing. Materials & methods: This research presents a new label-free fluorescent aptasensor for ATP measurement that incorporates a DNA aptamer, SYBR Gold and single-walled carbon nanohorns. Results: The aptasensor showed selectivity toward ATP and a low limit of detection (37.6 nM). The linear detection range was 100–50,000 nM, and the fluorescence intensity and ATP concentration logarithm showed an excellent linear correlation (R2 = 0.9924). Conclusion: The developed aptasensor may be used to detect cellular ATP in cancer cells and could be employed for biological sample analysis. The benefits of the aptasensor, such as its simplicity, speed, cost–effectiveness, specificity and sensitivity, give it promising implications as a potentially adaptable sensing platform.