The emotional victory came a day after two members of the Ukrainian Olympic team pulled out of the Games in support of demonstrators back home after widespread anti-government protests left dozens dead and hundreds injured.
The Ukraine delegation held a minute's silence at the Olympic village on Wednesday in the presence of National Olympic Committee chief Sergei Bubka.
"Personally, I had a meeting with the girls and the federation's president because we needed to share our knowledge and show our support," Bubka told reporters.
"I gave them my personal thoughts and personal advice. I told them 'it will be your day'."
So it proved in a tense race after which Ukrainian team members were overcome by emotion.
"When I was on the podium I couldn't stop crying. I tried to calm down and was trying to hide it behind my skis. They were tears of happiness, not only mine, but of the whole country, our team," Valj Semerenko told reporters.
"We are so happy that the people of Ukraine are happy back home and that something good happened for our country," said Olga Pidhrushna.
"Many people wrote to us: 'girls, please show everyone in Ukraine what you can do'. It would be not right from us not to start today," she later added in a news conference before which a minute's silence was respected.
"I hope now all the people in our country may be happy, including those who are fighting at the Maidan (Independence Square)."
Norway took the bronze after Ukraine's Pidhrushna lost ground on the final of four 6km legs to Russian Olga Vilukhina before rallying to cross the line 26.4 seconds ahead.
The capacity crowd roared in delight as Vilukhina had Pidhrushna in her sights before the final shooting section.
The Russian made a mistake, though, while the Ukrainian biathlete shot clean.
Russia had won the last two women's relays at the Olympics, but did not seem too disappointed.
"This silver medal is like gold for us because we won it at home," Olga Zaitseva told reporters.
"This is the result of many years of hard work, fighting against those people who did not believe in us."
France, who won silver in Vancouver four years ago, retired after Marie-Laure Brunet felt weak during her opening leg.
"It was a panic thing. She could not catch her breath. But she walked into the medical centre under her own steam," coach Siegfried Mazet said.
Ukraine's only other Winter Olympic gold medal was won by figure skater Oksana Baiul in the women's individual event at the Lillehammer Games in 1994.
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