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Sesame seed oil cake (SSOC) was applied as a novel substrate for production of high-added value pullulan by Aureobasidium pullulans KY767024 (a melanin-deficient strain). The optimization of production conditions by central composite design showed that the maximum production yield (54.50 g pullulan/kg substrate) was obtained under optimum conditions (Liquid-solid ratio (LSR) of 1.7 v/w, inoculum size of 7 mL, and temperature of 23 °C), which was confirmed by the experimental data (52.50 ± 0.73 g/kg). The obtained pullulan had an average-molecular weight of 358.736 kDa. The present peaks in the FT-IR spectrum validated that the obtained purified precipitate was composed of pullulan. Also, the XRD pattern and TGA analysis showed that the produced pullulan had a fully amorphous structure with high thermal stability. In addition, the pullulan solutions had a different flow behavior in different concentrations and with an increase in concentration, their behavior changed from Newtonian to pseudoplastic.