Abstract: Astaxanthin has a variety of physiological activities, but its most common form in nature is astaxanthin ester bound with different fatty acids. Hydrolysis of astaxanthin ester into free astaxanthin is an important research direction to improve the absorption and utilization of astaxanthin ester and to prepare astaxanthin ester with specific configuration. In this study, we heterologously expressed Sv-lip5 derived from Streptomyces violascens ATCC 27968 in Bacillus subtilis to explore the enzymatic properties and its application in astaxanthin ester hydrolysis. The results show that the optimal temperature of Sv-lip5 was 45 ℃; the optimal pH was 10.0 (Gly-NaOH buffer); and the enzyme activity of Sv-lip5 was higher than 55.7% after incubation at 35−55 ℃ for 21 h. As an alkaline resistant enzyme, the relative enzyme activity of Sv-lip5 could remain over 70% after incubation for 48 h under alkaline conditions. Sv-lip5 could hydrolyze astaxanthin ester in Haematococcus pluvialis oil effectively. Using p-nitrophenol palmitate as substrate, the specific activity of Sv-lip5 was 12.46 U·g⁻¹. The optimal hydrolysis conditions of astaxanthin ester were: pH of 8.0, ratio of ethanol to buffer of 1:12; and the optimal enzyme dosage and time were 900 mg and 12 h, respectively. After adding 900 mg enzyme powder to 5.5 mL reaction system, the hydrolysis effect was obvious in 1 h, and the highest hydrolysis rate was 98.27% at 12^th hour. The yield of astaxanthin in 200 μg astaxanthin ester was 147.48 μg. This method is high-efficiency for the biological hydrolysis preparation of free astaxanthin.