Viral gene vectors often rely on packaging cell lines, which provide the necessary factors in trans for the formation of virus-like particles. Previously, we reported on a first-generation packaging cell line for gene vectors, which are based on the B-lymphotropic Epstein-Barr virus (EBV), a human γ-herpesvirus. This 293HEK-derived packaging cell line harbors a helper virus genome with a genetic modification that prevents the release of helper virions, but efficiently packages vector plasmids into virus-like particles with transducing capacity for human B cells. Here, we extended this basic approach towards a non-transforming, virus-free packaging cell line, which harbors an EBV helper virus genome with seven genetic alterations. In addition, we constructed a novel gene vector plasmid, which is devoid of a prokaryotic antibiotic resistance gene, and thus more suitable for in vivo applications in human gene therapy. We demonstrate in this paper that EBV-based gene vectors can be efficiently generated with this much-improved packaging cell line to provide helper virus-free gene vector stocks with transducing capacity for established human B-cell lines and primary B cells.