Abstract:[Background] Galium spurium, one of the main noxious weeds in crop fields, is mainly controlled by chemicals, which causes serious harm to the human body and the environment. Microbial herbicides with precise targets and environmental safety have practical significance for agricultural modernization. [Objective] To screen out the non-polluting and harmless strains with strong weed-controlling activities and provide new strain resources for the development of microbial herbicides. [Methods] The strains were isolated and purified by tissue culture method. The herbicidal activity and selectivity of the isolate were determined by the spore suspension method. Internal transcribed spacer (ITS) and EF-1/EF-2 gene sequencing was used to identify the isolate. The phylogenetic tree was constructed in MEGA 7.0 to reveal the phylogenetic relationship of the strain. Finally, the environmental conditions suitable for growth and sporulation of the strain were analyzed based on colony diameter and mycelial dry weight combined with blood cell counting. [Results] A strain DT-08C was isolated and identified as Fusarium paeoniae. The incidence rate of DT-08C infection in G. spurium reached 47.22%–93.93%, and the strain was safe to broad bean, pea, maize, Chinese cabbage, tomato, cucumber, and pepper. The optimum media for colony growth and sporulation of DT-08C was PSA and OA, respectively. [Conclusion] F. paeoniae DT-08C is highly pathogenic to G. spurium but safe to broad bean, pea, maize, Chinese cabbage, tomato, cucumber, and pepper, and it has wide adaptability to the environment. The results provide strain resources for the further field application.