Technical Abstract: The purpose of this study was to prepare a novel temperature-sensitive spray gel containing silver nanoparticles and investigate its anti-bacterial properties in vitro. Methods: The aqueous complex gel was prepared by Pluronic F127 (18-22%) and Pluronic F68 (3-9%) through a cold method to obtain a proper gelation temperature at 37°C. Viscoelastic properties of the system were measured by rheological measurements and the physicochemical properties were evaluated by UV-vis spectrophotometer, MJ-22 Dia-reflex metaloscope, and Zetasizer Nano ZS90. Atomic Absorption Spectrometry (AAS) was used to assess the silver content in the nanoparticles. The in vitro antimicrobial activity was evaluated by disk diffusion test, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). Results: Temperature-sensitive spray gel containing silver nanoparticles with 20 wt% F127 and 6 wt% F68 aqueous solution had suitable fluidity at 25°C and was semi-solid at 37°C. The UV-vis absorption spectra indicated that a broad absorption peak centered at 420 nm. Silver nanoparticle sizes were mainly 50.9 nm. The gel achieved a modest viscosity value with the optimized formulation. Temperature-sensitive spray gel containing silver nanoparticles was shown to be an effective antibacterial agent and bactericide. Conclusions: Temperature-sensitive spray gel containing silver nanoparticles was an effective treatment for bacterial vaginosis (BV). This optimized silver nanoparticle dosage form showed great potential for further development in the treatment of BV.