In this work, a label-free fluorescence biosensor for simple detection of the HIV-1 gene was proposed by using toehold-mediated strand displacement reactions (TMSDRs) combined with a non-enzymatic target recycling amplification strategy. In this system, two TMSDRs were used. In the presence of the HIV-1 gene, an autocatalytic DNA machine can be activated. This leads to the generation of numerous free G-rich sequences, which can associate with a fluorescent dye N-methylmesoporphyrin IX (NMM) to yield an amplified fluorescence signal for the target detection. This sensing platform showed a high sensitivity towards the HIV-1 gene with a detection limit as low as 1.9 pM without any labelling, immobilization, or washing steps. The designed sensing system also exhibits an excellent selectivity for the HIV-1 gene compared with other interference DNA sequences. Furthermore, the presented biosensor is robust and has been successfully applied for the detection of the HIV-1 gene in a real biological sample with satisfactory results, suggesting that this method is promising for simple and early clinical diagnosis of HIV infection. Thanks to its simplicity, cost-effectiveness and ultrasensitivity, our proposed sensing strategy provides a universal platform for the detection of other genes by substituting the target-recognition element.