Aflatoxin B1 (AFB1) is the most toxic mycotoxin and is widespread in moldy feed. The use of biological removal methods to reduce AFB1 has become a research hotspot. This study aimed to isolate lactic acid bacteria (LAB) capable of removing AFB1 from moldy feeds and assessed the removal capacity under various environmental conditions. A strain named Lactobacillus brevis DN-1 was isolated from feed samples and showed 71.38% AFB1 percent removal. Furthermore, DN-1 showed good antifungal activity against Aspergillus flavus BNCC336156 and Aspergillus parasiticus BNCC335939. The optimum growth temperature and pH of DN-1 were 37 °C and 6.0, respectively, and DN-1 grew well in the concentration range of 0–20 µg/L AFB1. Under a temperature of 20–40 °C, pH of 3.0–9.0, and anaerobic conditions, the percent removal of AFB1 was more than 60%. An analysis of the different components of DN-1 showed that cell wall adsorption was the main removal method and suggested the pathway for AFB1 removal by LAB. In addition, strain DN-1 was used as a biological preservative in artificially contaminated peanut and sunflower cakes, which significantly inhibited the growth of mold and production of AFB1. In brief, this study highlights the potential use of DN-1 as a preventive agent against aflatoxicosis via strong removal capability in the application of fermented feed or food.