As a reducing agent and stabilizer of silver ions, fiber is a green way to synthesize nano-silver materials. The dialdehyde cotton fabric was obtained by the selective oxidation of sodium periodate, and then cotton fabric loaded with silver nanoparticles (AgNPs) was synthesized through the in situ stabilization on oxidized cotton and reduction reaction of the oxidized cotton fabric cooperated with glucose in this paper. The structure and composition of resulted cotton fabric modified by AgNPs were characterized by scanning electronic microscope, energy dispersive spectrum of X-ray, X-ray diffraction and infrared spectrum etc, and the possible generation mechanism of AgNPs in the oxidized cotton was investigated. The influence factors on the load yield of AgNPs in oxidized cotton fabric were analyzed, and the antibacterial activity of nanosilver fabrics against S. aureus and E. coli was also detected. The results indicated that the load yield of 6.33% for AgNPs in the fabric was achieved while the aldehyde content of oxidized fabric was 0.089mmol/g, the molar concentration ratio of glucose vs AgNO3 was 2:1, the AgNO3 concentration was 0.05mol/L and the pH value of reaction solution was about 8. The spherical AgNPs reduced from Ag+ uniformly stabilized on the surface of the oxidized cotton fabric as both reducer and carrier, the crystalline size of AgNPs was about 28 nm, furthermore the coordinate actions were formed between the oxidized cotton fiber and AgNPs. The prepared nano-silver loaded cotton fabrics had the antibacterial rates of 99.97% and 99.99% against S.aureus and E.coil, respectively, and then the antibacterial rate after 50 washes was still above 97.53%, which indicated a great potential for antibacterial textiles and protection materials of fibers.