The chief aim of the present work is to investigate the preparation for silver nanoparticles antimicrobial agent and its antibacterial activity on cotton fabric. In this study, antimicrobial agent was fabricated by a reaction between an amino-terminated hyperbranched polymer (HBP-NH2) and silver nitrate solution of certain concentration. Meanwhile, cotton fabric was oxygenated to afford aldehyde groups which could connect with the amino groups of the HBP-NH2 to provide cotton fabric with durable antimicrobial properties. The effects of different biocides made of various molecular structures of synthetic material (HBP-NH2) on antimicrobial properties of nano-silver colloid solution were discussed, and three different agents generated then were characterized in following aspects as silver nanoparticles size and distribution by using DLS, TEM and UV-vis. Furthermore, the fabric structure, mechanical properties and antimicrobial property of treated cotton fabric were also tested. Included in this part of experiment were transverse micro morphology of cotton fibre by SEM, fabric strength retention after finishing, silver nanoparticles molecular conformation on fabric and content analysis via ICP-AES. The results showed that stable silver nanoparticles collide solution with 20-30 nano, applied on oxidized cotton fabric under certain condition, could produce ideal antibacterial rate over 94% of bacterial reduction to both Staphylococcus aureus (S.aureus) and Escherichia coli (E.coli) after 50 consecutive washings.