A sensitive and specific electrochemical biosensor was developed for quantitative detection of HIV-related DNA by integrating toehold strand displacement reaction (TSDR) with cruciform DNA crystal. The TSDR system can be specifically triggered by HIV-related DNA generating plentiful double-stranded DNA products and releasing the target DNA to undergo an additional cycle of amplification. The hybridization reaction of double-stranded DNA products with capture probes on Au electrode surface makes the binding sites of cruciform DNA crystal available. After the attachment of cruciform DNA crystal and the employment of streptavidinylated alkaline phosphatase, a significantly amplified electrochemical signal is obtained. This strategy was applied to the highly sensitive detection of HIV-related DNA in a linear range from 1 pM to 100 nM and a detection limit as low as 0.21 pM was achieved. The proposed strategy can be accomplished without the employment of any enzymes or intensive purification. In addition, this method has good selectivity and anti-interference ability, which could be applied in complex samples with comparable analytical performance. For these advantages, the proposed strategy holds great potential for rapid and early clinical diagnosis of HIV infection.