Kanae Kijima, 42, who has married twice since she was detained in 2009, killed three men in the space of eight months through carbon monoxide poisoning, by burning charcoal briquettes after giving them sleeping tablets.
A spokesman for Japan's top court confirmed it had ruled against an appeal lodged by Kijima.
Her legal team has claimed her innocence, saying the three men were likely to have committed suicide, according to public broadcaster NHK.
The death penalty has overwhelming public support in Japan, despite repeated protests from European governments and human rights groups.
Executions are by hanging, however it can take years before they are carried out.
The case has been closely followed in Japan and major media flashed news of the top court decision across television screens.
Kijima writes a blog from the detention centre where she has been held, detailing her life inside, the food and talking about men she likes.
In the latest post on Thursday, she wrote to her readers: "I hope to see you again somewhere someday."
Kenzo Ando, 80, died in his home in Chiba prefecture in May 2009, and three months later 41-year-old Yoshiyuki Oide was found poisoned in a rented car, also from briquette fumes.
Kijima was convicted without the witness testimony or confession often relied upon in Japanese prosecutions.
Instead prosecutors rested their case on layers of circumstantial evidence, such as Kijima's purchases of sleeping pills and coal briquettes, in addition to the fact that she had met with each man shortly before he died.
She was also found guilty of seven other lesser crimes, including fraud and theft.
In another "black widow" case, Chisako Kakehi, 70, is awaiting trial in June for allegedly killing several men.
(This story has not been edited by NDTV staff and is auto-generated from a syndicated feed.)