Objective The novel μ opiate receptor (MOR) antagonists with biological activity was searched based on computer aided drug design method. Methods The MOR was employed as the target protein, and the Glide module of Schrodinger software was used to virtually screen the numerous compounds included in ZTNC-15 open source compound database. According to the molecular docking score, skeleton structure, binding mode and compound acquisition, one compound that may be as an antagonist was selected. The anti-fentanyl-induced acute death model of mice and the experiment of improving fentanyl-induced respiratory depression of rats were used to evaluate the biological activity of A₆ against MOR. Finally, molecular dynamics simulation method was employed to analyze the possible mechanism of MOR-A₆ interaction. Results The molecular docking score of compound A₆ was comparable to that of naloxone. The results of animal experiments showed that the LD₅₀ value of fentanyl-induced death of mice in A6 prevention administration group [13.2(95%CI 12.0-14.5) mg/kg] was 1.3 times and higher than that in model group [10.5(95%CI 9.6-11.5) mg/kg], and the mortality of mice in experimental group was significantly lower than that in model group (P<0.05); Compared with model group, the carotid oxygen saturation (SaO₂) of rats in A₆ prevention administration group increased significantly at 15, 20 and 25 min after fentanyl injection (47.91%±3.17% vs. 62.63%±4.14%, 52.99%±3.92%vs. 69.57%±3.17%, and 58.16%±2.45% vs. 77.10%±4.93%, P<0.05). Molecular dynamics simulation of A₆ and MOR showed that ASP147 amino acid residue of A₆ is consistent with the predicted results of molecular docking, and has a large contribution on binding free energy. Conclusions A novel compound A₆ has obtained by virtual screening which could effectively antagonise fentanyl-induced acute toxic death in mice and improve fentanyl-induced respiratory depression in rats. The mechanism may be related to the hydrogen bond formation of ASP147 amino acid residue with MOR.