Efficient removal of medical wastewater and pharmaceutical production wastewater containing Beta-blockers is currently the focus and difficulty of research. In this study, the UASB reactor was used to achieve efficient removal of Beta-blockers. The removal rate of atenolol, metoprolol and propranolol is about 80%, 60% and 60%, respectively. The relevant functional microorganisms were Thermotogae, Anaerolineae and Bacteroidetes. Beta-blockers interfered with the metabolism of bacteria and methanogens, resulting in reduction of methane production and accumulation of acetic acid, which in turn affected the performance of UASB reactor. Metoprolol, atenolol and propranolol are mainly removed by anaerobic co-metabolism with starch, of which oxidative degradation is the main route. This study provides a new idea for the complete and efficient biodegradation of Beta-blockers wastewater.