Based on the helix4-exchanged HPV16 L1 and HPV18 L1, HPV16 L1 Bi and HPV18 L1 Bi, we have successfully realized the controlled hybrid-assembly of HPV16/18 L1 Bi VLPs (bihybrid-VLPs) in vitro. The bihybrid-VLPs were further confirmed by fluorescence resonance energy transfer (FRET) and complex-immunoprecipitation (Co-IP) assays. The ratio of 16 L1 Bi and 18 L1 Bi in bihybrid-VLPs was verified to be 3:5 based on a modified magnetic Co-IP procedure, when mixing 1 equiv pentamer in assembly buffer solution, but it changed with conditions. In addition, the bihybrid-VLPs showed identical thermal stability as that of normal VLPs, suggesting high potential in practical applications. The present study is significant because it modified one of the vital steps of virus life cycle at the stage of virus assembly, supplying a new approach not only to deepen structural insights but also a possibility to prepare stable, low-cost, bivalent antivirus vaccine. Furthermore, the controlled hybrid-assembly of bihybrid-VLPs in vitro provides suggestions for the design of effective multivalent hybrid-VLPs, being a potential to develop broad-spectrum vaccines for the prevention of infection with multiple types of HPV.