Abstract: The skin of Takifugu obscurus is high in collagen. There is a growing trend to study the preparation method and functional properties of fish skin collagen due to the additive values. This research aimed to optimize the collagen extraction processing from the skin of dark-striped pufferfish including the effects of pepsin enzyme addition, enzymatic hydrolysis time, ultrasonic power and ultrasonic time. The dextran gel column chromatography method was used to separate and identify the extracted and salt-desorized collagen. The inhibitory activity of extracted collagen on tyrosinase and hyaluronase was studied. Results showed that, the best extraction rate of collagen was 71.6%±2.7% at pepsin 4.2%, enzymatic hydrolysis time 38 h, ultrasonic power 320 W, and the ultrasonic time 9 mins. The inhibition rate of tyrosinase and hyaluronidase was 85% and 90% respectively, at concentration of Takifugu obscurus skin collagen was 0.8 mg/mL and the pH was 5.5, which performed better than the commercial fish skin collagen. This study indicated that the skin collagen of Takifugu obscurus had the great potential in whitening and anti-inflammatory application, which could be further applied in the field of cosmetics.