In this work, π‐conjugated block copolymers consisting of poly(phenyl isocyanide) (PPI) and polyfluorene (PF) segments are facilely prepared by one‐pot sequential polymerization of phenyl isocyanide (monomer 1) and 7‐bromo‐9,9‐dioctylfluorene‐2‐boronic acid pinacol ester (monomer 2). The Pd(II)‐terminated PPI is first prepared via polymerizing monomer 1 catalyzed with phenyl alkyne‐Pd(II) complex and then utilized to initiate the controlled Suzuki cross‐coupling polymerization of monomer 2, yielding various PPI‐b‐PF copolymers possessing controlled molar mass and narrow dispersity. Owing to the helical conformation of PPI segment and π‐conjugated structure of PF segment, PPI‐b‐PF copolymers present distinctive optical property and fascinating chiral self‐assembly behavior. During the self‐assembly process, chirality transfer from helical PPI block to the supramolecular aggregates of helical nanofibers occurs to afford optically active helical nanofibers with high optical activity. Furthermore, the self‐assembled helical nanofibers exhibit excellent circularly polarized luminescence performance.