Objective To establish a method for preparing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudoparticles (SARS-CoV-2 pps). Methods The optimized sequence of spike (S) gene of SARS-CoV-2 was designed, synthesized and used to construct the mammalian cell expression plasmid. The resultant plasmid was used to transfect 293T cells, and the expressed S protein was detected using immunofluorescence. The S expression plasmid was further used to transfect 293T cells together with lentiviral genome backbone based pseudoparticles package plasmids containing enhanced green fluorescent protein (EGFP) reporter gene. The supernatant of 293T cells was collected and used to infect Vero E6 cells and Huh7 cells. The intracellular expression of EGFP was observed. The confirmed SARS-CoV-2 pps was used to infect Vero E6 cells, and then we observed the effects of membrane fusion inhibitors chloroquine and arbidol hydrochloride, monoclonal antibodies to S1 protein of SARS-CoV-2 and convalescent sera of patients with coronavirus disease 2019 on the pseudoparticles infection. Results The 293T cells transfected with SARS-CoV-2 S plasmid could react with monoclonal antibodies to S1 protein of SARS-CoV-2 and convalescent sera. SARS-CoV-2 S plasmid and human immunodeficiency virus (HIV) pseudoparticles package plasmid were used to transfect 293T cells together, adding the supernatant to Vero E6 and Huh 7 cells, the intracellular expression of EGFP was observed at 36 h and 72 h, respectively. Compared with Vero E6 cells, there were more EGFP-positive cells for Huh7 cells. Two membrane fusion inhibitors, one human monoclonal antibody against SARS-CoV-2 S1 protein and two convalescence sera could effectively inhibit the infection of SARS-CoV-2 pps of Vero E6 cells. Conclusion We have established a method for preparing SARS-CoV-2 pps, which can be used for the anti-SARS-CoV-2 drug screening and vaccine evaluation.