The firefly luciferase system is the most extensively utilized bioluminescence system in the field of life science at the moment. In this work, we designed and synthesized a series of alkene-conjugated luciferins to develop new firefly bioluminescence substrates, and further evaluated their activities in vitro and in vivo. It is worth noting that the maximum biological emission wavelength of novel luciferin analogue AL3 ((S,E)-2-(6-hydroxy-5-(3-methoxy-3-oxoprop-1-en-1-yl)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid) is 100 nm red-shifted compared with D-luciferin, while that of analogue AL4 ((S,E)-2-(5-(2-cyanovinyl)-6-hydroxybenzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid) is 75 nm red-shifted. The new substrate AL2 ((S,E)-2-(6-hydroxy-7-(3-methoxy-3-oxoprop-1-en-1-yl)benzo[d]thiazol-2-yl)-4,5-dihydrothiazole-4-carboxylic acid) showed better bioluminescence performance in vivo.