Abstract <p><i>Cry</i><i>pto</i><i>coc</i><i>cus neoformans</i>, an opportunistic fungal pathogen, can induce central nervous system infections, posing a life-threatening risk and imposing substantial global health challenges and economic burdens. Given the significant reduction in mortality achieved through early monitoring of <i>C. neoformans</i>, there is an urgent demand for a rapid detection method for this pathogen. Herein, we developed a rapid, sensitive, and specific assay for the detection of <i>C. neoformans</i> based on a one-pot recombinase polymerase amplification (RPA) and CRISPR/Cas12a system, which can be read using a real-time fluorescent PCR instrument or lateral flow strips. This assay exhibits high sensitivity, with a detection limit of 1 copy/µL for <i>C. neoformans</i>, and no cross-reactivity was observed across different fungal strains. Notably, the assay can be performed in harsh environments without reliance on complex equipment, making it suitable for point-of-care testing (POCT). Collectively, this method not only provides a robust alternative for <i>C. neoformans</i> detection but also offers valuable insights for the identification of other fungal pathogens.</p> Key points <p>• <i>We developed a one-pot RPA and CRISPR/Cas12a assay to detect</i>&#xa0;<i>Cryptococcus neoformans</i>.</p> <p>• <i>This assay exhibits high sensitivity and specificity</i>.</p> <p>• <i>Detection results can be obtained by three ways which is suitable for&#xa0;POCT</i>.</p> Graphical Abstract <p></p>