Aspergillus flavus is one of widely distributed fungal, which infects crops related foodstuffs and feeds, especially maize and peanut, upon cultivation, storage, manufacture, and transportation. A. flavus produces a lot of toxic secondary metabolites, and among them aflatoxins (AFs), recognized as Group 1 carcinogens, are the most important ones. AFs contamination severely affects food safety, brings huge economic losses and trade disputes. More importantly, AFs have high toxicity, high carcinogenicity, mutagenicity, and immune suppression, which severely threaten human and animal health. Therefore, the biosynthesis, regulation, prevention of AFs are the critical and hot points of the research of food safety. This study clarified the AFs biosynthesis process and its related regulatory mechanism, introduced various effective prevention methods, emphasized the effects of environmental factors (light, temperature, water activity, nutrient, pH value, oxidative stress) on AFs biosynthesis, illustrated different regulators involved in AFs regulation, and introduced diverse strategies including physical, chemical, and biological methods for AFs control and prevention, summarized the prevention strategies for AFs contamination, and prospected the key research fields, prevention and control measures in the future.