This study clarified biochemical overripeness characterization of ovulated eggs of Anguilla japonica and suggested a method maintained overripeness after ovulation for high hatching rates. In maturated Japanese eel eggs, the relationships between fertilization rate and hatching rate, and fertilization and survival rates were measured. DNA contents showed the significantly low 0.653 pg/ug protein in 20% downward hatching rate trial with decrease of hatching rate(P<0.05), whereas RNA/DNA ratio showed the significantly high 1.058 in 20% downward hatching rate trial(P<0.05). And activities of total alkaline protease and ACPase according to the hatching rate groups did not show the significant difference(P>0.05). The protein contents were assayed the significantly high 186.16 ug/mg protein in 20% downward hatching rate trial(P<0.05). However, the overripened eggs had lowed hatching rate, because of stimulate the overripening of normal maturated eggs due to the continuous supplement of protein (vitellogenin). We suggested that need to reduce supplement speed or interception of vitellogenin produced in live for prevent overripeness of maturated eggs after ovulation