Display omitted •Periphytic biofilm could remove As(III) from wastewater.•Calcite and the OH and CO groups in biofilm facilitate As(III) removal.•The BPS achieved stable and effective removal of As(III).•The As(III) adsorption process by biofilm obeys pseudo-second-kinetic model. A biochar and periphyton-based system (BPS) comprising of a biochar column and a periphyton bioreactor was designed to avoid the toxicity issue associated with removing As(III) from wastewater. Results showed that the periphyton can grow when As(III) is less than 5.0mgL−1. The BPS obtained a high As(III) removal rate (∼90.2–95.4%) at flow rate=1.0mLmin−1 and initial concentration of As(III)=2.0mgL−1. About 60% of the As(III) was pre-treated (adsorbed) in the biochar column and the removal of the remaining As(III) was attributed to the periphyton bioreactor. The As(III) removal process by periphytic biofilm in the initial stage fits a pseudo-second-kinetic model. The calcite in the periphytic biofilm surfaces and the OH and CO groups were responsible for the As(III) removal. This study indicates the feasibility of the BPS for As(III) removal in practice.