Background and Objectives: Drug resistance in HIV-1 is one of the main causes of failure of antiretroviral therapy. Phenotypic detection of drug-resistant HIV-1 can provide guidance in selecting the optimal treatment regimen. Traditional phenotype assays are labor intensive and time consuming. Thus, a rapid and convenient phenotype assay with a single cycle of replication was developed and used in this study. Methods: Two restriction endonuclease sites, ApaI and AgeI, were inserted into the plasmid pSG3,DΔenv using site-directed mutagenesis. The reverse transcriptase and protease genes of HIV-1 were amplified from patients and cloned into the modified pSG3Δenv. Sixteen original recombinant pseudoviruses were generated. The phenotypic susceptibility of these 16 recombinant pseudoviruses to 12 antiretro viral drugs was determined using a luciferase reporter system, and the phenotype and genotype results were compared. Results: A modified phenotype assay with a single-cycle system was established, and its reproducibility and feasibility were validated. Approximately 89% of the phenotype results were in agreement with the genotype results; this slight disagreement may have been due to complex and multiple resistance mutations. The phenotype results showed that individual pseudoviruses with four thymidine analog mutations (TAMs) [M41L, T67N, L210W, and T215Y] in combination with various other mutations had different levels of resistance to nucleoside reverse transcriptase inhibitors (NRTIs). Mutations E44A, T69D, and V118I influenced the pattern of resistance of TAMs. The level of resistance to non-NRTIs (NNRTIs) was also variable when different NNRTI-resistance mutations were combined. Conclusion: The single-cycle pseudovirus phenotypic susceptibility detection system reflects HIV-1 drug resistance, especially for complex resistance mutants, and could be used to screen new antiretroviral candidates.