CRISPR/Cas systems have become integral to nucleic acid detection and biosensors, offering high specificity and sensitivity. Various CRISPR/Cas systems, such as CRISPR/Cas9, CRISPR/Cas12, CRISPR/Cas13, CRISPR/Cas14, and CRISPR/Cas3, utilize different mechanisms to detect or differentiate biological activities and nucleotide sequences. These systems are often combined with techniques like PCR, LAMP, RPA, and transcriptional technologies for effective diagnostics. CRISPR/Cas-based biosensors have been developed to detect nucleic acids of viral and bacterial pathogens in clinical samples, with applications in biosecurity, food safety, and environmental assessment. They offer better specificity compared to other molecular diagnostic methods. This review covers various CRISPR/Cas-based nucleic acid detection methods, highlighting their development, components, and operational challenges. It also discusses the advantages and disadvantages of different systems, providing empirical suggestions for improvement. Key CRISPR/Cas systems include: 1. **CRISPR/Cas9**: Utilizes Cas9 protein and gRNA to recognize and cleave target sequences, often combined with amplification techniques. 2. **CRISPR/Cas12/14**: Recognizes and cleaves target sequences, with collateral cleavage activity, suitable for nucleic acid detection and other applications. 3. **CRISPR/Cas13**: Targets RNA sequences and has collateral cleavage activity, useful for nucleic acid detection. 4. **CRISPR/Cas3**: Contains helicase and endonuclease domains, capable of unwinding and cleaving dsDNA. The review details specific CRISPR/Cas-based methods, such as CRISPR/Cas9-based systems (e.g., NASBACC, CAS-EXPAR, FEUDA, CASLFA), CRISPR/Cas12-based systems (e.g., HOLMES, DETECTR, OR-DETECTR, CRISPR-ENHANCE), CRISPR/Cas13-based systems (e.g., SHERLOCK, CARMEN/Cas13a, FIND-IT), and CRISPR/Cas3/Cas14-based systems. Each system is evaluated based on its advantages, such as high sensitivity, specificity, and ease of use, as well as challenges like background noise, off-target detection, and the need for sample pretreatment. Overall, CRISPR/Cas-based nucleic acid detection systems offer significant advancements in molecular diagnostics, but their commercialization is hindered by operational complexities and the need for specific gRNA sequences.CRISPR/Cas systems have become integral to nucleic acid detection and biosensors, offering high specificity and sensitivity. Various CRISPR/Cas systems, such as CRISPR/Cas9, CRISPR/Cas12, CRISPR/Cas13, CRISPR/Cas14, and CRISPR/Cas3, utilize different mechanisms to detect or differentiate biological activities and nucleotide sequences. These systems are often combined with techniques like PCR, LAMP, RPA, and transcriptional technologies for effective diagnostics. CRISPR/Cas-based biosensors have been developed to detect nucleic acids of viral and bacterial pathogens in clinical samples, with applications in biosecurity, food safety, and environmental assessment. They offer better specificity compared to other molecular diagnostic methods. This review covers various CRISPR/Cas-based nucleic acid detection methods, highlighting their development, components, and operational challenges. It also discusses the advantages and disadvantages of different systems, providing empirical suggestions for improvement. Key CRISPR/Cas systems include: 1. **CRISPR/Cas9**: Utilizes Cas9 protein and gRNA to recognize and cleave target sequences, often combined with amplification techniques. 2. **CRISPR/Cas12/14**: Recognizes and cleaves target sequences, with collateral cleavage activity, suitable for nucleic acid detection and other applications. 3. **CRISPR/Cas13**: Targets RNA sequences and has collateral cleavage activity, useful for nucleic acid detection. 4. **CRISPR/Cas3**: Contains helicase and endonuclease domains, capable of unwinding and cleaving dsDNA. The review details specific CRISPR/Cas-based methods, such as CRISPR/Cas9-based systems (e.g., NASBACC, CAS-EXPAR, FEUDA, CASLFA), CRISPR/Cas12-based systems (e.g., HOLMES, DETECTR, OR-DETECTR, CRISPR-ENHANCE), CRISPR/Cas13-based systems (e.g., SHERLOCK, CARMEN/Cas13a, FIND-IT), and CRISPR/Cas3/Cas14-based systems. Each system is evaluated based on its advantages, such as high sensitivity, specificity, and ease of use, as well as challenges like background noise, off-target detection, and the need for sample pretreatment. Overall, CRISPR/Cas-based nucleic acid detection systems offer significant advancements in molecular diagnostics, but their commercialization is hindered by operational complexities and the need for specific gRNA sequences.