Systemic lupus erythematosus (SLE) is a chronic autoimmune disease, with anti-double-stranded DNA (anti-dsDNA) antibodies as its serological biomarkers. However, conventional anti-dsDNA antibody detection methods, which mainly rely on antibody-binding assays, often suffer from limited sensitivity and specificity, cumbersome procedures, and poor suitability for accurate clinical analysis. Herein, we developed an integrated detection system combining a circularly permuted fluorescent protein (cpFP)-based biosensor with a microfluidic chip for rapid and reliable anti-dsDNA antibody detection. The biosensor, cpR-dsAb-C1, was engineered from mApple by inserting an affinity peptide identified through phage display, enabling specific recognition of the variable region of anti-dsDNA antibodies. The biosensor exhibited good sensitivity, specificity, and anti-interference capability. Furthermore, integration of cpR-dsAb-C1 with a polydimethylsiloxane (PDMS)-based microfluidic chip yielded a microfluidic detection platform with good linearity for rapid antibody analysis. Clinical validation showed significantly higher anti-dsDNA antibody levels in patients with SLE than in healthy controls, and the results were consistent with those obtained using routine clinical methods, with an accuracy exceeding 95%. Overall, this system provides a promising low-cost, efficient, and accurate strategy for the early diagnosis and dynamic monitoring of SLE.