Background:　Streptococcus　agalactiae　or　group　B　Streptococcus　(GBS)　is　a　leading　infectious　cause　of　neonatal　morbidity　and　mortality.　It　is　essential　to　establish　a　robust　method　for　the　rapid　and　ultra-sensitive　detection　of　GBS　in　pregnant　women　with　premature　rupture　of　membrane　(PROM).　Methods:　This　study　developed　a　CRISPR-GBS　assay　that　combined　the　advantages　of　the　recombinase　polymerase　amplification　(RPA)　and　CRISPR/Cas12a　system　for　GBS　detection.　The　clinical　performance　of　the　CRISPR-GBS　assay　was　assessed　using　vaginal　or　cervical　swabs　that　were　collected　from　179　pregnant　women　with　PROM,　compared　in　parallel　to　culture-based　matrix-assisted　laser　desorption　ionization　time-of-flight　mass　spectrometry　(culture-MS)　method　and　real-time　quantitative　polymerase　chain　reaction　(qPCR)　assay.　Results:　The　CRISPR-GBS　assay　can　be　completed　within　35 min　and　the　limit　of　detection　was　as　low　as　5　copies　μL−1.　Compared　with　the　culture-MS,　the　CRISPR-GBS　assay　demonstrated　a　sensitivity　of　96.64%　(144/149,　95%　confidence　interval　[CI]　92.39–98.56%)　and　a　specificity　of　100%　(30/30,　95%　CI　88.65–100%).　It　also　had　a　high　concordance　rate　of　98.88%　with　the　qPCR　assay.　Conclusions:　The　established　CRISPR-GBS　platform　can　detect　GBS　in　a　rapid,　accurate,　easy-to-operate,　and　cost-efficient　manner.　It　offered　a　promising　tool　for　the　intrapartum　screening　of　GBS　colonization.　©　2023,　The　Author(s).