Bioluminescence (BL) imaging is among the most popular methods for visualizing biological processes in vitro, in live cells, and even in the whole organism. The alkylation of aminoluciferin at the 6'-amino site (2-hydroxyethyl luciferin, HE-AL) can emit more enhanced and sustained BL signals than d-luciferin and aminoluciferin, which is suitable for approaches that require long integration time such as three-dimensional animal BL imaging. Nevertheless, the drawback of HE-AL is that the synthesis procedure is complex, which leads to few BL probes based on HE-AL. In this Article, an efficient and facile approach to synthesize HE-AL was first established with an appreciate yield of 64% as compared with 15% previously reported. Then, designing HE-AL as a small molecular probe (R)-2-(6-((2-((2-((2,4-dinitrophenyl)thio)benzoyl)oxy)ethyl)amino)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid (DNPT-HS) was designed to detect endogenous hydrogen sulfide (H2S) in cancer cells and nude mice for further exploration of the biological roles of H2S in physiological and pathological processes. It was observed that DNPT-HS had excellent sensitivity in the luciferase-transfected cancer cells and nude mice, and the signal-to-noise ratios of DNPT-HS in cells and nude mice were 26 (Huh7 cells), 21 (MDA-MB-231 cells), and 7 (mice), which was the most efficient probe for imaging endogenous H2S. Overall, the excellent imaging properties of DNPT-HS for endogenous H2S make it a potentially powerful tool for further elucidating H2S biological functions.