Microcystin-LR (MC-LR) removals linked with empty bed contact time (EBCT), temperature and backwash strategies are important for biologically active filter (BAF) application but remain not entirely clear. In addition, there is still a lack of understanding about the bio-regeneration of MC-LR adsorption capacity in a BAF. This study examined MC-LR removals by a granular activated carbon-biologically active filter (GAC-BAF) as a function of EBCT, temperature, and backwash strategies. Results demonstrated that the optimum EBCT (1.2 h for this study) likely depended upon an optimum superficial velocity as well as the GAC column height. A superficial velocity not greater than 0.5 m/h was deemed appropriate according to this research. The MC-LR removals achieved at the optimum EBCT reached 54.8%–72.0%. A higher temperature resulted in a greater MC-LR removal; adsorption might contribute to effective MC-LR removal by the GAC-BAF at a lower temperature. A strategy (Air + air-water + water) was suggested as an optimal backwash strategy for GAC-BAFs in terms of MC-LR removal. A comparison of the effectiveness of the experimental GAC-BAF in continuous and discontinuous operations on MC-LR removals was conducted, from which indirect evidence for bio-regeneration of MC-LR adsorption capacity of the GAC was obtained.