Preparation of HIV-1 Env protein and development of ultrasensitive detection method of HIV-1 gp41 antibody are challengeable tasks for early diagnosis of patients with HIV virus. The env gene fragments were obtained from the plasmid with HIV-1 env by PCR, and were cloned into T vector, and then were sequenced. The linear env gene fragments were prepared by Rapid Translation System (RTS) Linear Template Kit, the Env proteins were obtained by RTS Kit, and were purified by magnetic bead method. The Env protein's bioactivity was identified by Western Blotting. The prepared Env proteins were labeled with CdTe quantum dots (QDs), and the goat anti-HIV-1 gp41 IgG antibodies were detected by CdTe QDs-labeled Env proteins, the fluorescent signals were recorded by photoluminescent spectroscopy. Results show that the prepared Env proteins own bioactivity, the CdTe QDs-labeled Env proteins can combine with anti-HIV-1 gp41 IgG antibodies, and formed the Env-gp41-CdTe QDs complex, the fluorescent intensity of final products were negatively associated with the concentration of anti-HIV-1 gp41 IgG antibodies in sample, the limitation of detection is 100 pg/mL. In conclusion, HIV-1 Env proteins with bioactivity were successfully prepared by RTS, established CdTe QDs labeled Env proteins-based fluorescent immunoassay can successfully detect HIV-1 gp41 IgG antibodies, which has great potential in early diagnosis of HIV.