Fusarium root rot in alfalfa (Medicago sativa L.) known as an important soil-borne disease, infects root crown and root tissues of plants in many regions of the world. A fungal strain, AD-G14was selected from 73 isolates from rhizosphere soil and root of healthy alfalfa as a potential biological control agent. It exhibited significant antagonistic effects against four Fusariumstrains that isolated from rotten alfalfa root. Based on morphology and the internal transcribed spacer (ITS) and 18S rDNA gene sequences, AD-G14 was identified as Actinomucor elegans. The sterile fermentation broth of AD-G14 was effective in inhibiting mycelial growth and spore germination of various Fusarium strains. Moreover, AD-G14 significantly increased the plant height and above-ground biomass of alfalfa that had been affected by root rot. The effectiveness of AD-G14 in controlling Fusarium root rot reached a significant rate of 43.97%. Inoculation with AD-G14 led to a significant increase in the activities of peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) and significantly decreased the malondialdehyde (MDA) content in alfalfa plants affected by Fusarium root rot. These findings indicate that AD-G14is effective in controlling Fusarium root rot in alfalfa and holds promise for development as a biocontrol agent.