A fascinating optically important 3-Hydroxyflavone derivative 2-(2,5-dimethoxyphenyl)-3‑hydroxy-4H-chromen-4-one was synthesized and its single-crystal structure was studied. This compound was used as a dopant and incorporated into the pullulan matrix by electrospinning technique. Functionalized pullulan nanofiber mat was found to possess dopant molecules organized in an X-aggregated fashion accounting for the twisted structure of the dopant molecules. The diameter of the nanofiber mat was reduced from 250 nm to 120 nm upon fabrication with dopant due to the nanofiber mat's increased crystallinity. Dopant molecules imparted absorption and emission properties into the fabricated nanofiber mat and turned it to be highly fluorescent triggering yellowish-green emission with a large Stokes shift of 193 nm due to X-aggregation. The improved water resistance of the fabricated pullulan nanofiber mat was witnessed by an increased water contact angle from 27.3° to 62.5° due to the incorporation of hydrophobic dopant molecules. The thermal and mechanical stability of the fabricated nanofiber mat was improved upon fabrication. The predicted absolute quantum yield was obtained in the 34.1- 40.3% range. The optimum indirect optical band gap of 2.25–2.50 eV and refractive index of 2.1–3.4 for the designed nanofiber mat suggested the potential application as OLED material.