It is well known that periphyton biofilm is a kind of micro-aggregates that ubiquitously distributed in natural aquatic bodies, usually attaching on the sediment surfaces as well as the surfaces of suspended materials in waters. Therefore, it is more practical and meaningful to study the migration and transformation of phosphorus among "sediments-periphyton biofilm-overlaying water" system rather than the commonly sediments-overlaying water system. In this study, the periphyton biofilms used in experiments were collected from a kind of artificial substrate that designed special for the fast enrichment of periphyton, and then the migration and transformation of phosphorus among "sediments-periphyton biofilm-overlaying water" system was investigated. Results showed that the presence of periphyton biofilm interface could significantly decrease the content of total phosphorus, dissolved total phosphorus, dissolved inorganic phosphorus in overlying water under both situations of aeration and non-aeration, and obviously reduce the release of exchangeable phosphorus from sediments to overlying water. This study shows that it should take the periphyton biofilm interface into accounts when studying the phosphorus behaviors between sediments and overlying water.