Based on microchip electrophoresis-chemiluminescence detection(MCE-CL) platform,a new method of MCE separation assisted double cycle chemiluminescence signal amplification was established for the detection of HIV gene(HIV-DNA),with horseradish peroxidase-labeled DNA(HRP-DNA) as signal probe,utilizing HRP to catalyze the CL reaction of luminol and hydrogen peroxide and the hybridization of target molecules with DNA,and combined with T7Exo enzyme-assisted signal amplification.In this method,two DNA single strands P1 and P2 firstly formed P1/P2 double-stranded complexes.They then formed a ternary complex when the target HIV-DNA existed,which could be degraded specifically by T7Exo to release the HIV-DNA and P2 single strands.The released HIV-DNA was rehybridized with the P1/P2 double-stranded complex,and the T7Exo continued to degrade the complex.While the released P2 single strand was hybridized with HRP-DNA portion to form P2/HRP-DNA double strand,which was degraded by T7Exo,simultaneously releasing HRP-DNA fragment and P2 single strand.The released P2 sequence was rehybridized with HRP-DNA,which was degraded by T7Exo to produce a large number of HRP-DNA fragments.High sensitive detection of target molecules could be realized by double cycle signal amplification.Under optimized experimental conditions,the logarithmic values of HIV-DNA concentration in the range of 1.0×10-14-5.0×10-9 mol/L showed a good linear relationship with the chemiluminescence intensity of the system.The detection limit(S/N=3) was 1.6×10-15 mol/L.At four spiked levels of 0.10,0.25,1.0,10(×10-12 mol/L),the recoveries range from 93.0%-103%,and relative standard deviations(RSDs) was 0.50%-3.7%.The proposed method was applied to the analysis of HIV-DNA in human serum with satisfactory results.