Two kinds of surface modification methods, chemical oxidation--surface covered with iron ion and chemical oxidation--surface grafting of gelatin, were used to improve the surface properties of polyethylene bio-carrier, and the composition and microbial species in biofilm formed on the modified bio-carrier surface were investigated. The experimental results showed that two kinds of modification methods could effectively increase the content of biofilm extracellular polymeric substances. The extracellular polysaccharide contents were increased by 63% and 18.5%, respectively, which benefited the biofilm adhesion and formation. The extracellular protein contents were increased by 43% and 15.4%, which indicated that biofilm activity was greatly increased. Microscopy observation showed that the appearance of metazoan in the modified biofilm was earlier than in the unmodified biofilm, which could be indicative for high efficiency and stable performance. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis showed that the composition of microbial species on the biofilm had little significant difference before and after modification, with the similarity of microbial species of 98.0% and 93.8%, respectively, but the quantity of dominant genus and microbial concentration were greatly increased.