The resource utilization of food waste is crucial, and single-cell protein (SCP) is attracting much attention due to its high value. This study aimed to convert food waste to SCP by Yarrowia lipolytica. It was found the chemical oxygen demand (COD) removal rate 77 ± 1.70% was achieved at 30 g COD/L with the protein content of biomass only 24.1 ± 0.4% w/w biomass dry weight (BDW) in one-stage fermentation system. However, the protein content was significantly increased to 38.8 ± 0.2% w/w BDW with the COD removal rate 85.5 ± 0.7% by a two-stage fermentation process, where the food waste was firstly anaerobically fermented to volatile fatty acids and then converted to SCP with Yarrowia lipolytica. Transcriptomic analysis showed that the expression of SCP-producing genes including ATP citrate (pro-S)-lyase and fumarate hydratase class II were up-regulated in the two-stage transformation, resulting in more organic degradation for SCP synthesis.