This study aims to develop a positively charged nanofiltration (NF) hollow fiber membrane for effective removal of ciprofloxacin from water. A novel NF membrane was fabricated by hyperbranched polyethyleneimine (PEI) induced cross-linking on a polyamide-imide hollow fiber support. The spongy-like, fully porous membrane support provides minimal transport resistance and sufficient mechanical strengths for water permeation under high pressures. It is found that the PEI modification significantly influences NF performance through the mechanisms of size exclusion, charge repulsion, and solute-membrane affinity. Specifically, after PEI induced cross-linking, the membrane pore size is significantly reduced. The membrane surface becomes more hydrophilic and positively charged. As a result of these synergic effects, the rejection of ciprofloxacin is substantially enhanced. Furthermore, experimental results show that the molecular weight of PEI has tremendous effect on NF performance of the as-modified membrane. The NF membrane modified by a high molecular weight PEI_60K exhibits the highest rejection, the lowest fouling tendency, and keeps a constant flux over the whole pH range. This study may have great potential for developing high-performance antifouling NF hollow fiber membranes for various industrial applications.