This paper describes a disposable and accurate aptasensor—that functions with signal amplification—for prostate-specific antigen (PSA) analysis in neutral (pH 7.4) condition. Combining aptamer-immobilized magnetic nanoparticles with rolling circle amplification (RCA) has provided a PSA detector with adequate sensitivity. In the absence of PSA, a portion of the complementary DNA-primer (cDNA-primer) bound with the aptamer and became the primer for the RCA reaction. The product of RCA contained G-rich bases that bound specifically with methylene blue (MB), thereby providing a measurable signal. In the presence of PSA, the aptamer would bind to it and fold, leading to fewer bound cDNA-primer units. Because the number of RCA primer elements decreased, the signal of MB weakened. The difference in the currents of MB measured in the presence and absence of PSA reflected the concentration of PSA. The intensity of the signal had a linear relationship with the logarithm of the PSA concentration over the range from 100 fM to 10 nM. The correlation of determination (R 2 ) of this calibration curve was 0.993, with a limit of detection of 22.3 fM.