Astaxanthin is a kind of carotenoid with a high antioxidant capacity, which has promising applications in feed and human health. As consumers demand more natural products, the biological production of astaxanthin has gained great attention. In this study, an astaxanthin producing Xanthophyllomyces dendrorhous strain DW6 was first isolated, whose astaxanthin synthetic pathway was determined by whole genome sequencing. Furthermore, fermentation parameters including temperature, pH and carbon and nitrogen sources used for the yeast growth and astaxanthin synthesis were also comprehensively investigated. Accordingly, a two-stage pH fermentation strategy was developed to relieve the metabolic stress and promote the astaxanthin accumulation. Transcriptome analysis found that the key genes of fada (encoding acetyl-CoA acyltransferase) for fatty acid degradation and GGPS1 (geranylgeranyl diphosphate synthase) for astaxanthin synthesis, as well as genes involved in the glutathione and peroxisome metabolism played major roles in the improved astaxanthin synthesis used in the two-stage pH process. Finally, an inferior substrate of sugarcane molasses was used as the sole carbon and nitrogen source, and 374.3 mg L⁻¹ of astaxanthin was produced with an astaxanthin content of 9.0 mg g⁻¹, representing the highest astaxanthin production from organic wastes by using X. dendrorhous. Overall, this study will pave the way for the large scale production of astaxanthin.