In this work, we report a new in-line method to tailor the surface properties of porous ultrafiltration membranes in a one-step spinning process. A highly charged polyimide P84 hollow fiber membrane can be obtained by dissolving as less as 3% of sulphonated poly(ether ether ketone) (SPEEK) in the bore liquid. The zeta-potential of the lumen surface of this new P84/SPEEK membrane is independent of pH in the range 4.5–10 and around three times higher than for pure P84 hollow fiber membrane (−58 mV vs. −21.5 mV). The P84/SPEEK membrane has uniform sponge-like structure and water permeance of 6.5 l/(m2 h bar).Filtration of a mixture of proteins with similar molecular weights: bovine serum albumin (BSA, MW ≈66.4 kDa) and hemoglobin (Hb, MW ≈64.5 kDa), but differing in electrostatic properties, through the pure P84 and P84/SPEEK membranes demonstrates an improved separation performance of the P84/SPEEK hollow fiber. In fact, this membrane separates the binary mixture of BSA and Hb at the highest selectivity of around 200 at pH 7.5. Moreover, at the same pH the flux decline for the P84/SPEEK membrane is significantly reduced in comparison to the pure P84 membrane.Surprisingly, surface charge modifications can be introduced during the coagulation process by adding polyelectrolytes to coagulation bath. This will offer new opportunities for in-line membrane modification during the spinning process.