The result on a snowy night when much of the world's attention was focused on political developments elsewhere in the Ukrainian capital, also eliminated the Italian club, who are now confined to the bottom of Group B.
Dynamo picked up the tempo after an uneventful first half and they were rewarded late in the second period, which was played in a heavy snowstorm at Kiev's Olympic stadium.
Greece defender Traianos Dellas put the Ukrainian champions ahead in the 73rd minute when he inadvertently headed into his own goal while trying to clear Tiberiu Ghioane's cross.
Substitute Maxim Shatskikh sealed victory for the home side nine minutes from time to the delight of some 55,000-strong home fans who had braved the foul weather to attend the match.
Shatskikh, who came on for Latvia striker Maris Verpakovskis just a few minutes earlier, tapped into an open goal after a nice feed from Brazilian midfielder Diogo Rincon.
Roma defender Giuseppe Scurto compounded the misery for the visiting side when he was sent off in the 76th minute for his second yellow card.
The Italians were missing several of their top players, including joint top scorer in Serie A Vincenzo Montella, through injury and suspension. Roma captain Francesco Totti was also absent due to a family bereavement.
Dynamo coach Josef Sabo said his team fully deserved their victory. "Roma is not only Totti, they have several outstanding players and their team is well balanced," Sabo said.
Earlier on Tuesday, more than 100,000 people poured into the city's main square, about a kilometre from the stadium, for the second straight day to protest against the results of Ukraine's presidential election.
Dynamo are top of Group B with 10 points, two ahead of both Real Madrid and Bayer Leverkusen, who drew 1-1 in the Spanish capital. Roma are out of contention with one point from five matches.
Roma coach Luigi Del Neri said it would be difficult to motivate his team for their final game at home to Real Madrid.
"I think we were less motivated than our opponents tonight but still played well, especially in the first half," he said.
"The team is gradually getting better and better, but anyway, we are Roma and we can't let ourselves down by playing badly against Real."
In their Group B opener two months ago, Dynamo were awarded a 3-0 win over Roma when Swedish referee Anders Frisk was injured by a coin thrown by a spectator at Rome's Olympic Stadium.