In order to solve the problem of high cytotoxicity in vitro of nano-silver antibacterial gel, and the problem of large nano-silver particle size and size distribution, this study prepared nano-silver antibacterial gel with better biocompatibility and good antibacterial effect by using physical cross-linking method and using poloxamer as dispersant when prepared nano-silver. In this study, nano-silver was prepared by photo-initiator method and by adding poloxamer as a dispersant, and then UV-visible absorption spectrum test and scanning electron microscopy (SEM) test were carried out using prepared nano-silver mixture and particles after drying respectively. The gel was prepared through adjusting its pH value by using sodium bicarbonate, and then pH value test, SEM test for cross-section of gel, swelling ratio test, viscosity test, inhibition zone test and in vitro cytotoxicity test were carried out. The test results showed that the maximum absorption wavelength of prepared nano-silver, using poloxamer as dispersant and ultra-pure water as solvent, was 414 nm, and the average nano-silver size was about 60 nm. The prepared nano-silver using poloxamer as dispersant had smaller particle diameter and narrower particle size distribution than those using PVP as dispersant. Similarly, the prepared nano-silver using ultra-pure water as solvent also had smaller particle diameter and narrower particle size distribution than those using distilled water as solvent. The pH value of the prepared gel was between 5.8~6.1. The dried gel section had many holes. The water absorption of gel was fine and the viscosity of gel was fit to coat on the gauze. In addition, the prepared gel with nano-silver had greater ability to inhibit Escherichia coli and Staphyloccocus aureus at the concentrations of 24, 18 and 12 μg/mL. And the biocompatibility of the prepared gel with nano-silver was good when the concentration below 24 μg/mL. Based on the above features, the nano-silver antibacterial gel could be used in the treatment of burn or other wounds.