This study investigated the adsorption and degradation characteristics of rhodamine B(RhB) by periphyton with or without NaN3, under a group of the dosage of periphyton(dry mass 0.1, 0.2, 0.3, 0.4 g/L), temperatures(10, 15, 20, 25, 30, 35 ℃), pH values(4, 6, 8, 10) and the initial mass concentration of RhB(1, 5, 10, 20, 40, 80, 160 mg/L). This study also investigated the degradation mechanism of RhB by periphyton with HPLC-MS detection. The results showed that periphyton had some functions in biosorption and degradation of RhB. Within the dosage of periphyton in this experiment, the removal rate towards RhB increased gradually with the raise of periphyton. The removal procedure of RhB by periphyton included two-stage synergistic processes, involving biosorption and biodegradation, with biosorption as the dominant process. The optimal pH and temperature for removal of RhB by periphyton without NaN3 were 6 and 30 ℃, respectively; the optimal pH and temperature for adsorption of RhB by periphyton with NaN3 were 6 and 20-30 ℃, respectively. The isotherm was fitted by the Langmuir model and the Freundlich model, meanwhile, the Langmuir model was more suitable in explaining the adsorption isotherm of RhB by periphyton with NaN3. The results of HPLC-MS detection showed that the degradation of RhB might produced an intermediate product with a molecular weight of about 391 and the degradation process involved a gradual deethylation.