Nanoluciferase (NLuc) is the emerging commercially available luciferase considering its small size and superior bioluminescence performance. Nevertheless, this bioluminescence system has some limitations, including narrow emission wavelength and single substrate. Herein, a series of novel furimazine derivatives at the C-6 and C-8 positions of the imidazopyrazinone core have been designed and synthesized for extension of the bioluminescence substrates. It should be noted that two compounds, molecules A2 (2-(furan-2-ylmethyl)-6-(4-(hydroxymethyl)phenyl)-8-(phenylthio)imidazo[1,2-a]pyrazin-3(7H)-one) and A3 (2-(furan-2-ylmethyl)-6-(4-amino-3-fluorophenyl)-8-(phenylthio)imidazo[1,2-a]pyrazin-3(7H)-one), display reasonable bioluminescence properties for in vitro and in vivo biological evaluations. In particular, compound A3 can broaden the application of NLuc bioluminescence techniques, especially for in vivo bioluminescent imaging.