Enrichment of chemically resistant hydrophobic polymers with polar biomolecules is relevant to the production of fiber-based drug delivery devices and adsorptive filtration media, as well as fibers for selective molecular recognition of antibodies, enzymes and nucleic acids. Polysulfone (PSU) is an amorphous polymer possessing high-strength, rigidity and excellent thermal stability. The preparation of PSU spinning solutions requires lengthy dissolution times at elevated temperature that tends to degrade commixed polar biomolecules. Using the highly polar metabolite creatinine, as a model system, a variety of co-solvents was evaluated for electrospinning commixed solutions of PSU and creatinine at room temperature. The selection of solvent systems was informed by Hansen solubility parameters. A binary system of N, N-dimethylacetamide (DMAc):methanol (4:1) was not found to be a suitable solvent because of the need for elevated temperature (80℃) to facilitate dissolution, and a binary solvent system of N, N-dimethylformamide (DMF):dimethyl sulfoxide (DMSO) (3:2) resulted in nozzle blockage during spinning. A binary system of DMAc:DMSO (13:7) enabled preparation of PSU with creatinine at ambient temperature, and sub-micron fibers substantially free of beads were produced continuously via electrospinning, yielding fiber diameters in the range 470–870 nm. The presence of creatinine was confirmed by high performance liquid chromatography (HPLC), and fiber morphology was examined by scanning electron microscopy (SEM).