Quinoxaline compounds are widely used in the fields of medicine and chemical industry, especially in the development of anticancer drugs, due to their significant biological activity. 6-fluoro-4-hydroxy-3-oxo-3,4-dihydroquinoline-1 (2H)-carboxylic acid tert-butyl ester was synthesized through a four-step reaction method in this paper, and its single crystal was obtained by solution crystallization method. Crystallographic analysis indicates that the compound belongs to monoclinic crystal system with space group C2/ c, the unit cell dimensions are a = 1. 286 63(10) nm, b = 2. 252 49(17) nm, c = 1. 015 64(7) nm, Z = 8, ρc = 1. 359 g·cm - 3 , R = 0. 053 8, Rw = 0. 140 6. The optimal structure was calculated using density functional theory (DFT) in B3LYP/ 6-311 + G (2d, p) mode. The results are basically consistent with those obtained by X-ray single crystal diffraction. The antitumor activity study shows that the compound has good antitumor effect. In addition, the electrostatic potential and frontier molecular orbitals of the molecules were calculated by DFT to further understand their physical and chemical properties.