Silver nanoparticles were synthesized in water-in-oil microemulsion using silver nitrate solubilized in the water core of a microemulsion as source of silver ions, ascorbic acid solubilized in the water core of another one as reducing agent, isooctane as the continuous phase, and sodium bis (2-ethylhexyl) sulfosuccinate as the surfactant. The influence of AgNO3 concentration, ascorbic acid concentration and the molar ratios of water to surfactant (W) in microemulsionon on the silver nanoparticles have been investigated by TEM, UV-Vis spectra and Zetasizer Nano. As a result, the silver nanoparticles possessed good dispersivity, and their diameters were about 10 nm when the concentration of AgNO3 was 0.2 mol / L, the concentration of ascorbic acid was 0.2 mol / L and the water core radius (W) was 10. The antibacterial property of the silver nanoparticles indicated the E.coli was completely killed when the concentration of the silver nanoparticles was 10 μg/mL.