[en] A rolling circle amplification chemiluminescence immunoassay (RCA-CLIA) was developed for precise quantitation of Aβ in plasma. Capture antibodies conjugated with magnetic beads and detection antibodies with collateral single-stranded DNA (ssDNA) were bound to Aβ42/Aβ40 antigens to form a typical double-antibody sandwich structure. The RCA reaction was triggered by the addition of ssDNA, which generated products with a large number of sites for the binding of acridinium ester (AE)–labeled detection probes, thereby realizing the purpose of the amplification. The RCA-CLIA method had higher sensitivity than conventional CLIA without loss of specificity. Under optimum conditions, the linear range of Aβ42 and Aβ40 detection was 3.9–140 pg/mL and 3.9–180 pg/mL, respectively, with corresponding low detection limits of 1.99 pg/mL and 3.14 pg/mL, respectively. Plasma Aβ42 and Aβ40 were detected in the blood of 21 AD patients and 22 healthy people, wherein this ratio could significantly distinguish AD patients from healthy individuals with a sensitivity of 90.48% and specificity of 63.64% for a cutoff value of 154. The Aβ42/Aβ40 ratio of plasma acts as an accurate indicator for AD diagnosis; therefore, detection of plasma Aβ using the RCA-CLIA exhibits great potential in noninvasive diagnosis and progressive assessment of AD.