In this study, the experiments employed two kinds of reduced microbial, Acidiphilumcryptum JF-5 and sulfate reducing bacteria (SRB), which can reduce Fe³⁺ and SO₄^2- in acid mining drainage (AMD) respectively. When reduction was done, they were mixed to form nano FeS, and then it was coated onto the particle limestone to improve the capability of removing arsenic through limestone permeable reactive barriers (PRBs). Batch adsorption experiments were conducted to explore the static adsorption mechanism of As(Ⅴ) and column experiments were performed for dynamic adsorption and migration of As(Ⅴ) in FeS-coated limestone. Some conclusions were reached: ① The coated biological FeS was of nanometer size and assumed crystalline form, which effectively improved the surface area of the limestone and the capacity for adsorbing arsenic. Infrared absorption spectrum showed that the main adsorption mechanism was chemical adsorption. ② The maximum adsorption amount of limestone coated with bio-nano FeS, was 187.46 μg/g, which was 30 times that of the adsorption amount of pristine limestone (6.64 μg/g). ③ The retentive capability of JF-5 and SRB coated limestone was better than that of the SRB and Fe(Ⅱ), and both of them were much better than the retentive capability of pristine limestone.