The CRISPR/Cas system consists of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas). The system forms an adaptive immune system in archaea and bacteria. The inherent defense mechanism enables these microorganisms to protect themselves against the invasion of foreign genetic material. The system functions of immune response including three main stages: adaptation, expression/maturation, and interference, each stage needs specific Cas proteins encoded by Cas gene located near the CRISPR sequences, along with other auxiliary proteins. In 2015, Zhang et al. reported Cas12a (Cpf1) as a member of the Class II type V CRISPR/Cas12a system, which possesses endonuclease activity. This finding holds great promise for its application in the field of biotechnology. In 2018, Doudna’s team first applied the CRISPR/Cas12a system for detecting HPV nucleic acid. The system comprises the following essential components in vitro detection: Cas12a, the crRNA sequence complementary to the target DNA, the PAM sequence, and the ssDNA reporter. Cas12a possesses a typical RuvC domain, displaying a canonical bilobed architecture that consists of a recognition (REC) lobe and a nuclease (NUC) lobe. The REC lobe contains the REC1 and REC2 domains, and the NUC lobe includes RuvC, PAM-interacting (PI), Wedge (WED), and bridge helix (BH) domains. The mature crRNA for Cas12a has a length of 42-44 nt, consists of repeat sequence (19/ 20 nt) and spacer sequence (23-25 nt). The crRNA spacer sequence has been found to require a length of 18 nt to achieve complete cleavage activity in vitro. Additionally, mutation in the bases of crRNA can indeed affect the activity of Cas12a. The PAM sequence plays a critical role in the recognition and degradation of DNA by the CRISPR/Cas system, enabling the system to distinguish between self and non-self genomic materials. Cas12a can effectively target the spacer sequence downstream of a T-rich PAM sequence at the 5" end. LbCas12a and AsCas12a both recognize the PAM sequences of 5"-TTTN-3", while FnCas12a recognizes the PAM sequences of 5"-TTN-3". All of these PAM sequences are located upstream on the non-template strand (NTS) at the 5" end. Cas12a (Cpf1), guided by the crRNA, binds to the target DNA by recognizing the PAM sequence. It exhibits the ability to induce arbitrary cleavage of ssDNA within the system while cleaving the target ssDNA or dsDNA. According to this feature, an array of nucleic acid detection methods has been developed for tumor detection and infection diagnostics, such as the DETECTR (RPA-CRISPR/Cas12a method) and HOLMES (PCR-CRISPR/Cas12a method) in 2018. Then, in 2019, Cas12aVDet (one-step detection method), where Cas12a protein was immobilized on the upper wall of the reaction tube. This not only prevented contamination from opening the tube but also reduced the detection reaction time. In 2021, the dWS-CRISPR (digital warm-start CRISPR) was developed as a one-pot detection method. It serves as an accurate approach for quantitatively detecting SARS-CoV-2 in clinical specimens. With the innovation of scientific technology, the high-sensitivity signal transduction technology has also been integrated with the CRISPR/Cas12a system, enabling direct detection of nucleic acids, and eliminating the need for nucleic acid amplification steps. Here, we elaborated the detection principles of CRISPR/Cas12a in in vitro detection. We discussed the different stages leading to the catalytic pathway of target DNA, and the practical applications of Cas12a in nucleic acid detection. These findings revealed a target interference mechanism that originates from the binding of Cas12a-guided RNA complex to complementary DNA sequences within PAM-dependent (dsDNA) regions. The crRNA-DNA binding activates Cas12a, enabling site-specific dsDNA cleavage and non-specific ssDNA trans-cleavage. The release of Cas12a ssDNase activity provides a novel approach to enhance the sensitivity and specificity of molecular diagnostic applications. Before these CRISPR/Cas12a-based nucleic acid detection methods can be introduced into clinical use, substantial work is still required to ensure the accuracy of diagnosis. Nevertheless, we believe that these innovative detection tools based on CRISPR/Cas will revolutionize future diagnostic technologies, particularly offering significant assistance in pathogen infection diagnosis for developing countries with relatively poor healthcare conditions and high prevalence of infectious diseases.