<p>Conjugated polymers (CPs) are π-electron-conjugated intrinsic fluorescent materials exhibiting unique photophysical and biophysical properties useful in sensing, labeling, and cargo delivery. While the photophysical properties related to conjugation lengths are known, the effect of conjugation lengths on intracellular cargo delivery has not been investigated yet. This study examined the impact of varying conjugation lengths in poly(<i>p</i>-phenylene ethynylene)s (PPEs) on intracellular protein delivery by evaluating protein loading capacity, serum stability, cellular entry pathway, and the amounts of proteins delivered into a model cancer cell line. While the protein loading, hydrodynamic diameter, and surface charge of all PPEs/protein complexes were similar regardless of backbone conjugation lengths, a fully conjugated PPE with a conjugation length of ~ 12 showed the highest intracellular protein delivery than PPEs containing shorter conjugation length than 12. The relatively poor delivery efficiency of other PPEs with a conjugation length of 9 or less was due to poor PPE/protein complex stability in the presence of serum, suggesting the critical role of backbone hydrophobicity. These findings signify the importance of conjugated polymer’s conjugation length in protein delivery efficiency, providing valuable insights into the design criteria for CP-based nanocarriers.</p> Graphical abstract <p></p>