Streptococccus agalactiae (S. agalactiae) is an important neonatal pathogen that is associated with mortality and morbidity. Therefore, we developed a rapid, accurate, and sensitive method based on multiple cross displacement amplification (MCDA) for the detection of the target pathogen. Four sets of MCDA primers were designed for targeting the S. agalactiae-specific groEL gene, and one set of MCDA primers with the optimum amplification efficiency was screened for establishing the S. agalactiae-MCDA assay. As a result, the newly-developed assay could be conducted at a fixed temperature (61°C) for only 30 min, eliminating the use of complex instruments. A portable and user-friendly nanoparticle-based lateral flow biosensor (LFB) assay was employed for reporting MCDA results within 2 min. Our results suggested that the detection limit of the S. agalactiae-MCDA-LFB assay is 300 fg per reaction, and no cross-reaction occurred with non-S. agalactiae strains. For 260 vaginal and rectal swabs, the detection rate of the MCDA-LFB assay was 7.7%, which was in accordance with the reference method of enrichment/qPCR, and higher by 4.6% than the CHROMagar culture. Moreover, the total procedure time of the MCDA-LFB assay was around 50 min, including sample collection, template preparation, MCDA reaction, and result reporting. Therefore, the MCDA-LFB assay is superior to enrichment/qPCR and CHROMagar culture and has great promise for point-of-care testing of S. agalactiae from vaginal and rectal swabs of pregnant women in resource-limited settings.