The study reports the synthesis, characterization, and computational analysis of (E)-4-((4-hydroxy-3-methoxy-5-nitrobenzylidene) amino) pyrimidin-2(1H)-one (C5NV), a compound derived from cytosine and 5-nitroaniline. The compound was synthesized through a simple condensation reaction and characterized using various techniques, including FT-IR, UV-Vis, NMR, and theoretical methods such as DFT and molecular docking. The structural characteristics of C5NV were optimized using the WB97XD/cc-pVDZ basis set, and vibrational frequencies were compared with experimental data. The electronic properties of C5NV in gas and solvent phases were investigated using TD-DFT, and its fluorescence properties were studied, showing three different wavelengths. Frontier molecular orbital (FMO) and molecular electrostatic potential (MEP) analyses were conducted, and non-covalent interactions were investigated using reduced density gradients (RDG). The molecular docking study between C5NV and the 2XNF protein showed a binding energy of −7.92 kcal/mol. The study provides insights into the physical, chemical, and biological properties of C5NV, highlighting its potential as a novel compound with interesting optical and biological characteristics.The study reports the synthesis, characterization, and computational analysis of (E)-4-((4-hydroxy-3-methoxy-5-nitrobenzylidene) amino) pyrimidin-2(1H)-one (C5NV), a compound derived from cytosine and 5-nitroaniline. The compound was synthesized through a simple condensation reaction and characterized using various techniques, including FT-IR, UV-Vis, NMR, and theoretical methods such as DFT and molecular docking. The structural characteristics of C5NV were optimized using the WB97XD/cc-pVDZ basis set, and vibrational frequencies were compared with experimental data. The electronic properties of C5NV in gas and solvent phases were investigated using TD-DFT, and its fluorescence properties were studied, showing three different wavelengths. Frontier molecular orbital (FMO) and molecular electrostatic potential (MEP) analyses were conducted, and non-covalent interactions were investigated using reduced density gradients (RDG). The molecular docking study between C5NV and the 2XNF protein showed a binding energy of −7.92 kcal/mol. The study provides insights into the physical, chemical, and biological properties of C5NV, highlighting its potential as a novel compound with interesting optical and biological characteristics.