Breath ammonia detection has been a significant non-invasive approach for disease diagnosis. However, the reported methods for breath ammonia detection suffer from high cost, time-consuming, complex operation, or low selectivity. Fluorescence analysis as an alternative method has not been introduced in the detection of breath ammonia due to its insufficient sensitivity. In this work, a rationally designed strategy is proposed to improve the sensitivity of fluorescence sensors by increasing the amount of recognition site and enhancing the brightness of the solid-state fluorescence in sensor molecule. Briefly, three curcumin (CUR) derivatives BFCUR, MBFCUR and MBFCURM with D-π-A-π-D conjugated system were synthesized. Compared with the other two control molecules MBFCUR and MBFCURM, whose phenol groups at one or both ends of BFCUR were protected by a methyl group, BFCUR has two response sites, thus, two intramolecular charge transfer (ICT) interactions occurred after exposure to ammonia, suggesting the improved sensitivity. Moreover, the large steric hindrance of boron trifluoride in BFCUR can prevent the π-π stacking of molecules and endow BFCUR with bright solid-state fluorescence. BFCUR was subsequently loaded into electrospun nanofibers (ENFs) with high specific surface area, which further improved the detection sensitivity. The obtained BFCUR-loaded ENFs sensor (denoted as BFCUR-ENF) exhibited ultra-low LOD of 22 ppb for ammonia. BFCUR-ENF is successfully applied to breath ammonia detection in healthy volunteers, patients with chronic kidney disease (CKD) and Helicobacter pylori (H. pylori) infection, demonstrating its great application potentials. Taken together, the first fluorescent sensor for breath ammonia detection was successfully constructed for noninvasive diagnosis of CKD and H. pylori.