Agro-industrial waste can be used to replace traditional carbohydrates, such as sucrose, starch, and glucose in many industrial fermentation processes. This study investigated the conversion of pre-treated waste sweetpotato vines (SV) into lipid by Trichosporon fermentans under the separate hydrolysis and fermentation (SHF) and the simultaneous saccharification and fermentation (SSF) processes. The results showed that SV autoclaving significantly increased the lipid accumulation of T. fermentans compared with acid or alkaline hydrolysis. The effects of different pre-treatments on SV were also studied by scanning electron microscopy and Fourier transform infrared spectroscopy, which showed the partial removal of the aliphatic fractions, hemicelluloses, and lignin during pre-treatment. Moreover, the lipid yield of T. fermentans in SSF was 6.98 g L-1, which was threefold higher than that (2.79 g L-1) in SHF, and the lipid contents of yeast in SSF and SHF were 36 and 25%, respectively. Overall, this study indicated that SSF using autoclaved SV could increase the growth and lipid production of T. fermentans and provided an efficient way to realize the resource utilization of waste SV.