Multi-gene-based approaches for tumor therapy have yet to be developed. Accordingly, we explored a combination treatment strategy by loading two kinds of therapeutic reagents simultaneously: pDNA and siRNA. Polyethylenimine-poly(l-serine) (PEI-PSer) obtained via ring-opening polymerization of l-serine N-carboxyanhydride initiated by polyethylenimines served as a potent cationic gene carrier. Proton nuclear magnetic resonance spectroscopy and gel permeation chromatography confirmed the successful preparation of PEI-PSer copolymers. The morphologies and effective binding capacities of PEI-PSer and genes were evaluated through gel retardation, scanning electron microscopy, and particle size and zeta potential assays. PEI-PSer displayed pDNA transfection and siRNA knockdown efficacy without affecting cell viability. The high gene transfer efficacy was mainly due to the excellent intracellular trafficking ability of PEI-PSer, as confirmed by confocal laser scanning microscopy and flow cytometry. Cell apoptosis assay further confirmed the combination antitumor effect aroused by the co-delivery of pKH-rev-casp-3 and siBcl2 by PEI-PSer. This study introduced a new carrier that delivers pDNA and siRNA simultaneously, thereby providing a potential strategy for tumor treatment.