We have investigated spin transport in films of polyfluorene, a π-conjugated polymer, using the technique of “spin pumping” from a ferromagnet substrate, namely Ni80Fe20 in a NiFe/polyfluorene/Pt trilayer device at room temperature. Pure spin current (without carrier injection) is generated in the polymer film by microwave excitation of the NiFe magnetic moment at ferromagnetic resonance conditions. The induced spin current through the ferromagnet/polymer interface crosses the polymer layer and is detected by a Pt overlayer in the device, where it is converted into electric current via the inverse spin Hall effect. We have successfully determined the spin diffusion length, λS, in the polyfluorene film by varying the polymer thickness in the trilayer structure, and found λS = 118 ± 9 nm. We also measured the charge-carrier mobility, μ in polyfluorene film using the time-of-flight technique and found it to be affected by dispersive transport. From the obtained λS and μ values, we estimated the spin relaxation time in polyfluorene to be ∼5 μs at room temperature.