Aflatoxin B1 is highly toxic, mutagenic, teratogenic, and carcinogenic and is a class I carcinogen. Peanuts, cotton, and corn may be affected by AFB1 during cultivation, which can seriously jeopardize human health. Developing a simple, sensitive, and selective method for detecting AFB1 is imminent. Aptamers are obtained through in vitro screening of ligands by single-stranded oligonucleotides (DNA or RNA) through exponential enrichment (SELEX) technology. As emerging highly selective recognition molecules, they have the advantages of strong affinity, good stability, and strong specificity. Because it does not have the function of signal conversion, it cannot produce physicochemical signals that can be detected in the process of specific binding with target molecules, so it is necessary to convert the process of specific binding of aptamers to target molecules into a process of easily detectable physicochemical signal changes. According to different conversion methods, aptamer biosensors are divided into electrochemical aptamer sensors, fluorescent aptamer sensors, colorimetric aptamer sensors, surface Raman-enhanced aptamer sensors, and so on. Herein, the recent progress and application of aflatoxin B1 detection by nucleic acid aptamer biosensors based on the above signals are reviewed, and the future development prospects and challenges of this kind of biosensor are summarized.