Immobilized bacterial agents (IBA) can increase the cell density and improve the environmental adaptability of bacteria. An estrogen-degrading bacterium, Novosphingobium sp. ARI-1, was immobilized in calcium alginate (CA) using an embedding method and applied to the removal of estrogens from natural sewage and cow dung. The optimum immobilization conditions were as follows: sodium alginate (SA) and CaCl·2HO concentrations of 5% (m/v) and 4% (m/v), respectively; a bacterial suspension to SA ratio of 1:2; and cross-linking for 6 h at 4°C. Immobilized strain ARI-1 mediated the biodegradation of estrone (E1), 17β-estradiol (E2), and estriol (E3) either individually or in combination and was tolerant of various temperatures and pH values. Immobilized ARI-1 removed 80.43%, 94.76%, and 100% of E1, E2, and E3 from sewage containing 1.75, 0.71, and 1.52 μg L of the three test estrogens within seven days, respectively. In cow dung containing initial E1, E2, and E3 concentrations of 0.71, 0.64, and 0.66 mg kg, respectively, E1 and E2 concentrations were below the limit of detection, and 1.09% of E3 remained after incubation with immobilized ARI-1 for seven days. These results confirmed the utility of immobilized strain ARI-1 for the removal of estrogens from environmental matrices.