Colloidal gold nanoparticles have been researched and utilized in many technical applications. However, the conventionalmethods to produce polyethyleneglycol (PEG) immobilized gold nanoparticles have to take several steps, including residual solvent removing. In the study, we propose an idea green route to synthesize gold nanoparticles by using Stenotrophomonas maltophilia in a one-pot reaction. The relationship between Au precursor and S. maltophilia was evaluated systematically. After PEG-SH addition, the bacterial cell wall was broken down and the synthetic nanoparticles could be released into culture medium. Furthermore, we identified that the crystal structure of synthetic gold nanoparticle was face-center cubic and PEG-SH was immobilized on synthetic gold nanoparticle ideally. The size of Au-PEG-SH was smaller than 30 nm. These findings suggest that gold nanoparticle with PEG-SH modification could be prepared in an eco-friendly one-pot reaction through the metabolic activity of S. maltophilia.