The extracellular polymeric substance (EPS) and surface properties of unsaturated biofilms of a heavy metal-resistant rhizobacterium Pseudomonas putida CZ1, in response to aging, pH, temperature and osmotic stress, were studied by quantitative analysis of EPS and atomic force microscope. It was found that EPS production increased approximately linearly with culture time, cells in the air-biofilm interface enhanced EPS production and decreased cell volume to cope with nutrient depletion during aging. Low pH, high temperature and certain osmotic stress (120 mM NaCl) distinctly stimulated EPS production, and the main component enhanced was extracellular protein. In addition to the enhancement of EPS production in response to high osmotic (328 mM NaCl) stress, cells in the biofilm adhere tightly together to maintain a particular microenvironment. These results indicated the variation of EPS composition and the cooperation of cells in the biofilms is important for the survival of Pseudomonas putida CZ1 from environmental stresses in the unsaturated environments such as rhizosphere.