Isothermal technology is an option for point-of-care diagnosis to replace PCR amplification. Recently, this approach has been updated into a simple, rapid, and cost-effective tool. The input can be defined as DNA, RNA, or certain artificial nucleic acids, and the output response can be an amplified signal or catalytic chemical reaction, allowing biosensing assays on various classes of biomolecules. The isothermal technologies used in the food safety detection can be classified into the following two categories: amplification and hybridization. Each category includes various methods designed to achieve a rapid and sensitive diagnose. Additionally, isothermal methods have made progress in the single-cell analysis due to the unbiased, linear nature of the amplification process. This review mainly describes specific isothermal detection techniques to give a better understanding of the method of choice when performing fast and simple point-of-care detection.