In biomedicine, rapid and sensitive nucleic acid detection technology plays an important role in the early detection of infectious diseases. However, most traditional nucleic acid detection methods require the amplification of nucleic acids, resulting in problems such as long detection time, complex operation, and false-positive results. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) systems have been widely used in nucleic acid detection, especially the CRISPR-Cas12a system, which can trans cleave single-stranded DNA and can realize the detection of DNA targets. But, amplification of nucleic acids is still required to further improve detection sensitivity, which makes Cas12a-based amplification-free nucleic acid detection methods a great challenge. This article reviews the recent progress of Cas12a-based amplification-free detection methods for nucleic acids. These detection methods apply electrochemical detection methods, fluorescence detection methods, noble metal nanomaterial detection methods, and lateral flow assay. Under various optimization strategies, unamplified nucleic acids have the same sensitivity as amplified nucleic acids. At the same time, the article discusses the advantages and disadvantages of each method and further discusses the current challenges such as off-target effects and the ability to achieve high-throughput detection. Amplification-free nucleic acid detection technology based on CRISPR-Cas12a has great potential in the biomedical field.