Abstract: In our previous work, the oleaginous yeast Rhodosporidium toruloides NP11 was employed as the chassis organism to construct an engineered strain, R. toruloides NA27. Through systematic optimization of fermentation conditions, strain NA27 achieved a total fatty acid titer of 95.4 g/L, while Nervonic acid (NA) accumulated to 44.2 g/L with a high volumetric productivity. As a representative lipid-accumulating yeast, R. toruloides is capable of synthesizing and storing substantial amounts of intracellular lipids. In addition, it has an endogenous cytosolic mevalonate (MVA) pathway that enables the biosynthesis of carotenoids, thereby allowing the concurrent production of lipids and carotenoids within a single fermentation process. Consistent with this metabolic capacity, the color of the yeast cells gradually intensified from light pink to deep red during fermentation, which closely correlated with the experimentally determined increase in carotenoid content. Given the intrinsic ability of the engineered strain to co-produce lipids and carotenoids, improving overall resource utilization efficiency while reducing downstream processing costs necessitates the development of an effective co-production and separation strategy. Accordingly, this study aimed to establish a simple and stable approach for the simultaneous separation and co-extraction of lipids and carotenoids from R. toruloides NA27. Fermentation broth obtained after 208 h of cultivation in a 5 L bioreactor was used as the feedstock. Initially, the effects of different cell harvesting methods on subsequent co-extraction performance were evaluated by comparing direct centrifugation for wet biomass recovery with flocculation-assisted harvesting using polymeric ferric sulfate and polyacrylamide as flocculants. Taking advantage of the pronounced differences in molecular structure, polarity, and physicochemical properties between lipids and carotenoids, three distinct separation strategies were designed and systematically evaluated for their suitability in lipid–carotenoid co-production. Among these strategies, a saponification-based approach was identified as the most effective and was therefore selected for further optimization. Single-factor experiments were conducted to optimize key operational parameters of the saponification-assisted co-extraction process, ultimately defining the optimal conditions for the simultaneous recovery of both products. Under the optimized conditions, the fermentation broth of engineered strain NA27 was directly processed, and the wet biomass obtained by centrifugation was used as the reaction substrate without any additional pretreatment or mechanical disruption of the yeast cell wall. Saponification was performed using a potassium hydroxide (KOH) solution at a concentration of 2 mol/L, with continuous stirring at 65℃ for 3 h. Isooctane was subsequently employed as the extraction solvent, and a ternary solvent system composed of water, absolute ethanol, and isooctane was applied to induce phase separation of the saponified products, thereby enabling efficient separation of a carotenoid-rich phase and a lipid-rich phase. The carotenoid phase was collected and concentrated by rotary evaporation to obtain crude carotenoids. The lipid phase was acidified with 2 mol/L hydrochloric acid (HCl), followed by two successive extractions with isooctane, and the combined organic phases were concentrated by rotary evaporation to yield crude lipids. The extraction efficiencies of lipids and carotenoids reached 74.9% and 53.0%, respectively. Notably, the saponification method achieved highly efficient separation of the two product streams, with no detectable lipid contamination in the recovered carotenoid fraction and a lipid selectivity as high as 99.4%. Overall, this study provides a technically feasible route and a sound theoretical basis for the industrial-scale co-production and simultaneous recovery of lipids and carotenoids from engineered R. toruloides, and offers valuable insights for the development of efficient downstream processing strategies in microbial multi-product biorefineries.