Abstract: Objective: To construct a highly sensitive isothermal nucleic acid detection method based on CRISPR Cas12a and evaluate its diagnostic ability for Cryptococcus neoformans in bronchoalveolar lavage fluid and cerebrospinal fluid. Methods: Conserved sequences of Cryptococcus neoformans were determined using NCBI Blast, and isothermal recombinase polymerase amplification primers and crRNA sequences were designed targeting these conserved sequences. Nucleic acids were extracted from standard strains of Cryptococcus neoformans and other strains (Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Staphylococcus aureus, Neisseria meningitidis, Streptococcus pneumoniae) to evaluate the specificity and the lowest detection limit of the method. Furthermore, bronchoalveolar lavage fluid and cerebrospinal fluid samples from 10 clinically diagnosed cases of Cryptococcus neoformans infection in the lungs and central nervous system were collected to evaluate the clinical detection performance of the nucleic acid diagnostic method constructed in this study. Results: A total of 6 primer and crRNA combinations were designed, and 1 optimal combination was selected after screening. The detection method constructed in this study exhibited high specificity (no cross-reaction with 7 other types of strains), a lowest detection limit of 5 copies/μL, and rapid detection process (completed within 1 hour). The positive detection rates for bronchoalveolar lavage fluid and cerebrospinal fluid clinical specimens from 10 cases each were 100%. Conclusion: The novel nucleic acid detection method constructed in this study has high specificity and a good detection limit, demonstrating excellent detection performance in clinical specimen testing, and showing strong potential for clinical application in the detection of Cryptococcus neoformans nucleic acids.