The unusual physio and chemical properties of gold nanoparticles are found to have more advantage in the field of medicine, diagnostics and biosensors. In the present document, it is reported that Justicia gendarussa leaf extract mediated synthesis of gold nanoparticles by the reduction of gold ions. Three different phytochemical fractions were prepared from methanolic leaf extract by liquid-liquid extraction method using immiscible solvents. The total polyphenols, flavonoids and electron donating capacity (DPPH assay) of each phytochemical fraction was analyzed. The 1mg/ml of diethyl ether phytochemical fraction produced more gold nanoparticles within 15 minutes when exposed to 10ml of 0.5mM chloroauric acid compared to chloroform and ethyl acetate phytochemical fractions. The structural characteristics of diethyl ether phytochemical fraction synthesized gold nanoparticles were characterized by UV-visible spectroscopy, Dynamic light scattering, Transmission electron microscopy, X-ray diffraction and Fourier transform-infrared spectroscopies. These biosynthesized gold nanoparticles showed surface plasmon resonance band at 536nm in UV-visible spectrum. The size of the gold nanoparticles ranged from 20 to 42nm and 62 to 88nm with spherical, triangle, truncated triangle and hexagonal shapes. From the Fourier transform-infrared spectra of diethyl ether phytochemical fraction and synthesized gold nanoparticles, the possible functional group involved in gold ions reduction and capping of gold nanoparticles were identified. The stability of gold nanoparticles for 5 month period and at different pH range (5-10) was analyzed by observing the changes in surface plasmon resonance of gold nanoparticles. Moreover, the diethyl ether phytochemical fraction showed no cytotoxicity up to 100μg/ml in RAW 264.7 cell line .