To evaluate effectiveness of enhancing and restocking stock of sea cucumber Apostichopus japonicus,it is necessary to find an effective and secure way to mark sea cucumber’s soft body wall which rejects external objects and heals up quickly.In this study,juveniles of sea cucumber(3.45±1.34) g were immersed in calcein(200 mg/L)for 24 h,and their ossicles were examined under Nikon 80i fluorescence microscope to judge calcein’s effectiveness.Then,influence of calcein concentration(normal concentrations including 50,200,400 mg/L;and high concentrations including 500,750,1 000 mg/L)and immersion time(8,24,48,72 h)on antioxidant enzymes SOD,CAT,and GSH-Px were studied to test its security.The results revealed that ossicles in upper layer of dermis of sea cucumber’s body wall,tentacle,podia and parapodia could be dyed by calcein,and showed green under fluorescence microscope with optical filter(λ:Ex 450-490 nm)7 days after being immersed in calcein solution.Brightness of stained ossicles was diversified because of immersion duration and ossicles’ maturation degree.In this study,both normal and high concentrations of calcein had no significant impact on activities of SOD and GSH-Px(P>0.05),while activity of CAT was restrained in 1 000 mg/L concentration treatment(P<0.05).Immersion duration had significant impact on activities of all three enzymes.Activity of SOD was significantly lower in the 72 h treatment than in other ones(P<0.05);activity of CAT declined with increasing immersion duration; and activity of GSH-Px in the 24 h treatment was significantly higher than in the 48 h one(P<0.05).Interaction between immersion duration and calcein concentration had significant impact on activities of SOD and CAT(P<0.05),and showed no significant effect on that of GSH-Px(P>0.05).The result indicated that calcein concentration lower than 750 mg/L and immersion time around 24 h influenced activity of all three enzymes less and marked ossicles effectively for examining.It is still necessary to take other factors such as oxygen into consideration for sea cucumber’s survival.