Sensitive detection of human immunodeficiency virus DNA (HIV-DNA) is essential for timely diagnosis and cure of the illness. Herein, a novel "enrichment-stowage-cycle" strategy was proposed to fabricate a multiple amplified electrochemiluminecence (ECL) biosensor for HIV-DNA detection. On the basis of the enrichment role of magnetic nanobeads, assembly role of copolymer nanospheres and strand displacement amplification (SDA), the processes were named as "enrichment", "stowage", and "cycle", respectively. The method employed electrochemiluminescent nanospheres (ENs) as signal labels by assembling three layers of CdSe/ZnS quantum dots (QDs) onto the surface of copolymer nanospheres. Compared to QDs, the same concentration of ENs can the enhance the ECL intensity by about 11.3-fold. SDA could further amplify the signals by about 3.77-fold, possessing high sensitivity for low-abundant biomarkers detection. The integration of magnetic separation improved detection specificity and stability, making the method possible for practical application. On the basis of magnetic separation, ENs and SDA, the ECL biosensor realized ultrasensitive detection of 39.81 fM HIV-DNA, which was more sensitive than other HIV-DNA analytical methods, with a wide dynamic range of 0.05 pM to 50 nM. The successful detection of HIV-DNA in complex samples with good sensitivity and accuracy indicated its potential utilization in early judgment of diseases and fabrication of signal amplification platforms.