[Objective] A microorganism from the samples of industrial spoilage with high-yield bacterium biofilm was isolated and identified. Meanwhile studies on characterization of biofilm formation were also carried out. [Methods] First of all, crystal violet stain was used to evaluate the ability of the biofilm-forming of screened strains in 96-cell microplates. And then, colonial morphology, physio-biochemical tests and 16S rRNA sequence analysis were adopted to determine the phylogenetic position of the isolated strains. Finally, the effects of attachment materials and culture temperature on the characterization of biofilm formation were also researched using scanning electron microscope (SEM) and crystal violet stain respectively. [Results] The best biofilm-forming strain was identified and clarified into Citrobacter werkmanii. And biofilm could be formed on the surfaces of glass, stainless steel and polyvinylchloride (PVC) respectively. Moreover the characterization of biofilm could be significantly influenced by temperature and the best biofilm was found on the PVC at 30 °C no matter which materials. [Conclusion] In industrial spoilage, the biofilm-forming strains could be found and its ability to produce biofilm could also be affected by materials and temperature.