Objective To develop a rapid and accurate assay for detection of Leishmania major based on the recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system, so as to improve the diagnostic efficiency of cutaneous leishmaniasis. Methdos Based on the internal transcribed spacer 1 (ITS1) gene sequence of L. major (GenBank accession number: KM052753.1) retrieved from NCBI as the target sequence, RPA primers, CRISPR RNA (crRNA), and single-stranded DNA (ssDNA) reporter probe were designed. An RPA-CRISPR/Cas12a assay was developed by screening RPA primers and optimizing the RPA reaction temperature. Genomic DNA was extracted from L. major, and the ITS1 gene sequence was amplified. The amplification product was cloned into the pUC18 plasmid to construct a recombinant plasmid, which was serially diluted to 8 concentrations of 2.3 × 10⁶, 2.3 × 10⁵, 2.3 × 10⁴, 2.3 × 10³, 2.3 × 10², 2.3 × 10¹, 2.3 × 10⁰, and 2.3 × 10⁻¹ copies/μl as templates for RPA-CRISPR/Cas12a assay and PCR assay to evaluate the detection sensitivity of the plasmid. L. major promastigotes were added to 200 μl of canine whole blood samples at a concentration of 1.8 × 10⁷ parasites/ml, and genomic DNA was extracted, and serially diluted into 4 concentrations of 1.8 × 10², 1.8 × 10¹, 1.8 × 10⁰, and 1.8 × 10⁻¹ parasites/ml as templates for RPA-CRISPR/Cas12a assay to evaluate the detection sensitivity of parasites. Genomic DNA from L. major, L. infantum, Toxoplasma gondii, Babesia gibsoni, Neospora caninum, and Anaplasma phagocytophilum were used as templates for the RPA-CRISPR/Cas12a assay to evaluate the detection specificity. Repeatability of the RPA-CRISPR/Cas12a assay was assessed by performing 5 independent experiments with 3 replicates each using the 2.3 × 10⁴ copies/μl recombinant plasmid as the template. To compare the detection performance of the RPA-CRISPR/Cas12a and the PCR assay, L. major promastigotes were added to 10 samples of 200 μl canine whole blood at a concentration of 1.8 × 10⁵ parasites/ml for RPA-CRISPR/Cas12a and PCR assays, with negative controls set simultaneously. Results The RPA-CRISPR/Cas12a method was established using the F7/R7 primer pair, with optimized reaction conditions set as 41 ℃ for 25 minutes. The limits of detection (LOD) of RPA-CRISPR/Cas12a and PCR assays were 2.3 copies/μl and 2.3 × 10³ copies/μl of the recombinant plasmid, and the LOD of the RPA-CRISPR/Cas12a assay was 1.8 promastigotes/ml of parasites; however, this assay showed no cross-reactivity of L. major with L. infantum, T. gondii, B. gibsoni, N. caninum, or A. phagocytophilum. Repeatability testing showed stable and consistent fluorescence intensities across different batches, with a relative standard deviation of 4.27% (< 5%). Both RPA-CRISPR/Cas12a and PCR assays accurately detected all 10 spiked canine whole blood samples containing L. major promastigotes, achieving the same detection rate and a 100% coincidence rate. Conclusion The RPA-CRISPR/Cas12a assay developed in this study is rapid, sensitive, specific, and reproducible for detection of L. major, which may provide reliable technical supports for diagnosis of L. major infections.