Abstract: In order to develop new, safe and efficient natural product herbicides, the weed harm in tobacco fields were investigated by the inverted "W" nine-point sampling method and by the three-layer and three-level visual observation method in Daozhen County and Yuqing County, Zunyi City, Guizhou Province, to clarify the dominant weed species and control needs. Aspergillus versicolor D5, which had significant herbicidal activity against dicotyledonous weeds in indoor pot experiments, was fermentated and extracted, and the active ingredients and contents were clarified. Then the herbicidal effect of D5 fermented extract was evaluated by stem and leaf spraying in the field. The results showed that:(1) There were 15 species of weeds belonging to 9 families in Daozhen tobacco fields. Dicotyledonous Asteraceae was the most diverse family, and Amaranthaceae was the dominant family as it had the highest relative abundance value of 84.2% in total weeds. A total of 39 weeds in 16 families occurred in Yuqing tobacco fields. There were 29 species, 9 species, and 1 species of weeds with hazard grade Ⅰ, Ⅱ and Ⅲ respectively. The dicotyledonous Polygonum was classified as hazard grade III. (2) Marine Aspergillus versicolor D5 fermented extract was eluted with 10% ethyl acetate-petroleum ether and then eluted with 100% ethyl acetate. There was basically no loss of active substances after concentration, including ST (14.6%) and DHST (0.4%). (3) Field trials found that, at a low dosage (675 g/ha), the fermented extract of D5 had a significant herbicidal effect, especially on dicotyledonous weeds, especially on Veronica didyma, with the fresh mass control effect of 61.4%. Meanwhile, the fresh mass control effect against Setaria viridis and Calystegia hederacea also reached more than 50%. Therefore, the fermentation extract of D5 has a broad anti-weed spectrum and significant herbicidal effect on dicotyledonous weeds, and has the potential to be developed into a new natural product herbicide.