Flow field-flow fractionation (FlFFF) is a technology to separate the molecules by size in an open channel. Molecules with different size have different diffusivities and are located vertically in different positions when passing through an open channel. In this study, hollow fiber membranes instead of conventional rectangular channels have been used as materials for the open channel and this change would decrease the cost of manufacturing. FlFFF is a useful technique to characterize the biopolymeric materials. Retention time, diffusion coefficients and Stokes radius of analysis can be calculated from the related simple equations. Hollow-fiber flow field-flow fractionation (HF-FlFFF) has been used for the characterization and separation of protein mixture in a phosphate buffer solution and has demonstrated the potential to be developed into a disposable FlFFF channel. The important indexes for the analytical separation are selectivity, resolution and plate height. The optimized separation condition for protein mixture of Ovalbumin, Alcohol dehydrogenase, Apoferritin and Thyroglobulin is ${dot V}_{out}/{dot V}_{rad}=0.65/0.85;mL/min$.