Due to the important pathological roles of the HIV-1 gp120, the protein has been intensively used in the research of HIV . However, recombinant gp120 preparation has proven to be difficult because of extremely low expression levels . In order to facilitate gp120 expression, previous methods predominantly involved the replacement of native signal peptide with a heterologous one, resulting in very limited improvement . Currently, preparation of recombinant gp120 with native glycans relies solely on transient expression systems, which are not amendable for large scale production . In this work, we employed a different approach for gp120 expression . Besides replacing the native gp120 signal peptide with that of rat serum albumin and optimizing its codon usage, we generated a stable gp120-expressing cell line in a glutamine synthetase knockout HEK293T cell line that we established for the purpose of amplification of recombinant gene expressions . The combined usage of these techniques dramatically increased gp120 expression levels and yielded a functional product with human cell derived glycan . This method may be applicable to large scale preparation of other viral envelope proteins, such as that of the emerging SARS-CoV-2, or other glycoproteins which require the presence of authentic human glycans.