There is a growing urgent need for point-of-care testing (POCT) devices that integrate sample pretreatment and nucleic acid detection in a rapid, economical, and non-labor-intensive way. Here, we have developed an automated, portable nucleic acid detection system employing microfluidic chips integrating rotary valve-assisted sample pretreatment and recombinase polymerase amplification (RPA)-T7-Cas13a into one-step nucleic acid detection. The RPA and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a were integrated into a single-chamber reaction. As a validation model, we used this method to detect Group B streptococci (GBS) DNA and achieved a detection sensitivity of 8 copies/reaction, which is 6 times more sensitive than gold-standard polymerase chain reactions (PCRs). Dual specific recognition of RPA with CRISPR/Cas13a makes our method ultraspecific, with correct detection of Group B streptococci from 8 kinds of pathogenic bacteria. For the 16 positive and 24 negative clinical GBS samples, our assay achieved 100% accuracy compared to the PCR technique. The whole procedure can be automatically completed within 30 min, providing a more robust, sensitive, and accurate molecular diagnostic tool for POCT.