The announcement comes eleven days after a naval clash that saw Russian forces open fire on and seize three Ukrainian warships near the Kerch Strait.
In a statement released on Thursday, the Pentagon said the timing of the flight - undertaken as part of the Open Skies Treaty, was “intended to reaffirm US commitment to Ukraine and other nations.”
It described the “unprovoked attack” in Kerch as part of “a pattern of increasingly provocative and threatening activity.”
Although the Open Skies Treaty gives each of the 34 state parties the right to gather information about the military forces and activities of other state parties, an extraordinary flight allows for countries to conduct the flights on a bilateral basis - hence the action by the US and Ukraine.
It is the first such flight since 2014, which came as part of the US response to Moscow annexing Crimea from Ukraine.
The Defense Department said US, Canadian, UK, German, French, Romanian and Ukrainian observers were aboard the aircraft during the observation flight. This flight was requested by the Arms Control Directorate of the Ukrainian General Staff.
A total of 25 US military personnel took part in the flight, including eight Defense Threat Reduction Agency personnel and a 17-person air crew from the 55th Wing out of Offutt Air Force Base, Nebraska.
Washington is said to be making preparations to sail a warship into the Black Sea, although the option may not be taken up.
The flight also comes days after Washington said it suspending its participation in a Cold War-era nuclear arms treaty with Moscow over violations of the deal by the Kremlin. Russia denies any such violations.
Russia has arrested and charged all 24 sailors that formed part of the Ukrainian flotilla involved in the clash, the youngest of whom is 18. They all stand accused of illegally entering the territorial waters of Russia.
Distress signals sent by the Ukrainian ships suggest they were fired on when sailing away in international waters.
Under the terms of a 2003 agreement between Russia and Ukraine, signed before the annexation of Crimea, the Kerch Strait and Azov Sea are considered shared waters.
Also on Thursday, Ukraine urged a gathering of dozens of foreign ministers to increase sanctions against Russia, accusing Moscow of sowing “instability and insecurity” across the West.
Speaking at a meeting of the Organisation for Security and Cooperation in Europe (OSCE) in Milan, Ukraine's foreign minister Pavlo Kilmkin denounced the seizure of the crew members.
“It is a matter of urgency to provide a prompt and consolidated international response to this act of aggression. Declarations are not enough. There must be action,” Mr Klimkin told the annual gathering.
“We must raise the cost for Russia with comprehensive and tailored sanctions," he added. "There can be no business as usual.”
However, Russian Foreign Minister Sergei Lavrov pinned the blame for years of escalating tensions on Ukraine and its allies - including the US.
“Striving for dominance, a small group of countries uses blackmail, pressure and threats,” Mr Lavrov said
“Kiev (is) free from any punishment, shielded by its Western sponsors, who justify all its outrageous actions,” he added.
Meanwhile, the Ukrainian parliament has voted to withdraw from a wide-ranging treaty on friendship with Russia.
The Supreme Rada overwhelmingly backed a motion by President Petro Poroshenko not to prolong the treaty when it comes up for renewal in April. Mr Poroshenko also announced a new dispatch of troops to the Russian border during a visit to a barracks.
Associated Press and Reuters contributed to this report