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To rapidly identify individuals infected with severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) and control the spread of coronavirus disease (COVID‐19), there is an urgent need for highly sensitive on‐site virus detection methods. A clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein (Cas)‐based molecular diagnostic method was developed for this purpose. Here, a CRISPR system‐mediated lateral flow assay (LFA) for SARS‐CoV‐2 was established based on multienzyme isothermal rapid amplification, CRISPR‐Cas13a nuclease, and LFA. To improve the limit of detection (LoD), the crispr RNA, amplification primer, and probe were screened, in addition to concentrations of various components in the reaction system. The LoD of CRISPR detection was improved to 0.25 copy/μl in both fluorescence‐ and immunochromatography‐based assays. To enhance the quality control of the CRISPR‐based LFA method, glyceraldehyde‐3‐phosphate dehydrogenase was detected as a reference using a triple‐line strip design in a lateral flow strip. In total, 52 COVID‐19‐positive and 101 COVID‐19‐negative clinical samples examined by reverse transcription polymerase chain reaction (RT‐PCR) were tested using the CRISPR immunochromatographic detection technique. Results revealed 100% consistency, indicating the comparable effectiveness of our method to that of RT‐PCR. In conclusion, this approach significantly improves the sensitivity and reliability of CRISPR‐mediated LFA and provides a crucial tool for on‐site detection of SARS‐CoV‐2.

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