AbstractInsect cells, especially Sf9 cells, are commonly used in biomanufacturing due to their advantages in high expression levels and post‐translational modification. However, the development of stable expression cell lines via random integration tended to be unstable. Site‐specific integration (SSI) is an alternative strategy. In this study, a φC31 ‐mediated cassette exchange system in Sf9 cells was established for SSI. The tagging cassette with the reporter gene egfp was randomly inserted into the cell genome. Potential platform cell lines were obtained by fluorescence‐activated cell sorting (FACS) and single‐cell cloning. Platform cell lines were selected by assessing the fluorescence expression, stability, and growth kinetics of cell lines. The selected platform cell lines were co‐transfected with the φC31‐containing plasmid and the targeting cassette. Green‐fluorescence‐negative clones were screened by hygromycin resistance and FACS. The resulting cell clones exhibited the expression properties of the platform cell lines. The rapid development of cell lines for the production of influenza subunit vaccines by the cassette exchange system demonstrated that the system constituted a versatile and reusable platform for the production of various recombinant proteins. Overall, the φC31‐mediated cassette exchange system in Sf9 cells has the potential to facilitate and accelerate biologics development.