The antioxidant and anticancer activities of solitary tunicate (Styela clava) hydrolysate manufactured with different proteases (Alcalase 2.4 L FG, Thermoase PC10F, and pepsin) and optimized by response surface methodology (RSM) were investigated. The hydrolysate produced by Alcalase generally had greater antioxidant and anticancer activities. Two major fractions, $F_1$ and $F_2$, were produced by applying Alcalase hydrolysates to Sephacryl S-100 HR gel filtration chromatography. Fraction $F_2$ with a lower molecular weight ($3.6{pm}0.1kDa$) had higher ABTS and DPPH radical scavenging activities than $F_1$ ($5.0{pm}1.0kDa$), with $IC_{50}$ values of 11.4 and $227.5{mu}g/mL$, respectively. Moreover, fraction $F_2$ had higher anticancer activitiy, with $IC_{50}$ values of 577.1, 1,163.3, and $887.2{mu}g/mL$ against AGS, DLD-1, and HeLa cells, respectively. Fraction $F_2$ was also rich in tyrosine, lysine, arginine, phenylalanine, and histidine. These results suggest that solitary tunicate hydrolysates have significant health-promoting effects with excellent antioxidant and anticancer activities.