Astaxanthin has high utilization value in functional food because of its strong antioxidant capacity. However, the astaxanthin content of Phaffia rhodozyma is relatively low. Adaptive laboratory evolution is an excellent method to obtain high-yield strains. TiO2 is a good inducer of oxidative stress. In this study, different concentrations of TiO2 were used to domesticate P. rhodozyma. Results showed that the optimal astaxanthin-producing P. rhodozyma JMU-ALE105 was obtained under domestication with 1000 mg/L TiO2 concentration on the 105th day. After fermentation, the astaxanthin content reached 6.50 mg/g, which was 41.61% higher than that of the original strain. The ALE105 strain was fermented by batch and fed-batch, and the astaxanthin content reached 6.81 mg/g. Transcriptomics analysis showed that astaxanthin synthesis pathway, fatty acid, pyruvate and nitrogen metabolism pathway of ALE105 strain were significantly up-regulated. Based on the nitrogen metabolism pathway, the nitrogen source was adjusted by ammonium sulphate fed-batch fermentation, which increased the astaxanthin content reached 8.36 mg/g. This study provides technical basis and theoretical research for promoting industrialization of astaxanthin production of P. rhodozyma.