Biological soil crusts (BSCs) are a useful tool for immobilization of metal(loid)s in mining areas. Yet, the typical functional microorganisms involved in promoting the fast development of BSCs and their impacts on arsenic(As) contaminated soil remain unverified. In this study, As-contaminated soil was inoculated with indigenous Chlorella thermophila SM01 (C. thermophila SM01), Leptolyngbya sp. XZMQ, isolated from BSCs in high As-contaminated areas and plant growth-promoting (PGP) bacteria (Bacillus XZM) to construct BSCs in different manners. After 45 days of ex-situ culture experiment, Leptolyngbya sp. XZMQ and bacteria could form obvious BSCs. Compared to single-inoculated microalgae, the co-inoculation of Leptolyngbya sp. XZMQ and Bacillus XZM increased soil pH and water content by 10% and 26%, respectively, while decreasing soil EC and density by 19% and 14%, respectively. The soil catalase, alkaline phosphatase, sucrase, and urease activities were also increased by 30.53%, 96.24%, 154.19%, and 272.17%, respectively. The co-inoculation of Leptolyngbya sp. XZMQ and Bacillus XZM drove the formation of BSCs by producing large amounts of extracellular polymeric substances (EPS). The three-dimensional fluorescence spectroscopy (3D-EEM) analysis showed that induced BSCs increased As immobilization by enhancing the contents of tryptophan and tyrosine substances, fulvic acid, and humic acid in EPS. The presence of the –NH2 and –COOH functional groups in tryptophan residues were determined using Fourier Transform Infrared Spectroscopy (FTIR). X-Ray Diffraction (XRD) analysis showed that there were iron (hydrogen) oxides in BSCs, which could form ternary complexes with humic acid and As, thereby increasing the adsorption of As. Therefore, BSCs formed by co-inoculation of Leptolyngbya sp. XZMQ and Bacillus XZM increased the immobilization of As, thereby reducing the content of soluble As in the environment. In summary, our findings innovatively provided a new method for the remediation of As-contaminated soil in mining areas.