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The scarcity of natural fossil fuels presents a promising opportunity for the development of renewable microalgae-based biofuels. However, the current microalgae cultivation is unable to effectively address the high costs of the production of biofuels. To tackle this challenge, this study focused on recruiting engineered Phaeodactylum tricornutum (FabG-OE) to enhance biomass accumulation and lipid production by employing food waste hydrolysate under temperature variations. The biomass and lipid accumulations of FabG-OE were improved effectively in mixed culture medium and food waste hydrolysate at a volume ratio (v/v) of 80:20 at 30 °C. It was found that oxidative stress might contribute to the overexpression of lipogenic genes, thereby leading to lipogenesis at 30 °C. Upscaling cultivation of FabG-OE at 30 °C using a semi-continuous strategy and batch strategy was conducted to achieve 0.73 and 0.77 g/L/d of biomass containing 0.35 and 0.38 g/L/d of lipid, respectively. In summary, these findings provide valuable insights for advancing microalgae-based biofuel production., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)