Developing eco‐friendly, nonirritant, low‐toxic, and high‐efficient surface active ingredients for detergents is an ongoing challenge in the detergent field. Surfactin is one of the representative lipopeptides produced by microorganisms. In this article, we report the surfactin isolated from cell‐free broth of Bacillus subtilis HSO121 and purified by reversed‐phase high‐performance liquid chromatography for detergent formulations. The biodegradability, acute dermal irritation, acute oral toxicity (LD50 and LC50), surface activity, washing efficiency, and compatibility with hard water of the purified biosurfactant surfactin have been studied to explore the feasibility for applications of the surfactin in detergents. Acute oral toxicity tests (LD50 > 5000 mg kg−1, LC50 > 1000 mg kg−1) and skin irritation tests (PII = 0) indicate that the surfactin is a low‐toxic and nonirritant ingredient for detergent formulation. Moreover, the surfactin shows excellent surface and interfacial properties of emulsification and wettability, high compatibility, and stability in a wide range of temperatures, pH, and hard water and acceptable properties in biodegradability and foaming ability, which suggests that the biosurfactant surfactin is a promising ingredient for detergent formations in our daily life and for industrial applications.