The legislation - which also includes aid for Israel and Taiwan and a potential ban on TikTok - comes after months of delay that saw Ukrainian forces run short of ammunition and suffer battlefield setbacks.
"I just signed into law the national security package that was passed by the House of Representatives this weekend, and by the Senate yesterday," Biden told reporters, saying he is "making sure the shipments start right away, in the next few hours."
"It's going to make America safer, it's going to make the world safer and it continues American leadership in the world and everyone knows it," he said of the legislation.
"It gives vital support to America's partners so they can defend themselves against threats to their sovereignty and to the lives and freedoms of their citizens."
Minutes after Biden spoke, the Pentagon announced a US$1 billion package for Kyiv using the new funding, including air defense munitions, artillery rounds, ammunition for HIMARS precision rocket launchers, anti-tank weapons and armored vehicles.
Ukraine's President Volodymyr Zelenskyy offered his thanks on social media Wednesday.
"I am grateful to President Biden, Congress, and all Americans who recognize that we must cut the ground under Putin's feet rather than obeying him, as this is the only way to truly reduce threats to freedom," he wrote.
Lengthy debate
Washington has announced new aid for Ukraine on just one other occasion this year, a US$300 million package in March that was only made possible by using money that the Pentagon had saved on other purchases.
The package Biden signed Wednesday - valued at a total of US$95 billion - only passed after months of acrimonious debate among lawmakers over how or even whether to help Ukraine defend itself against Russia's invasion, which Moscow launched in February 2022.
A similar aid package passed the Senate in February.
But it had stalled in the House of Representatives while Republican Speaker Mike Johnson - heeding calls from ex-president Donald Trump and his hardline allies - demanded concessions from Biden on immigration policies, before a sudden reversal.
The United States has been a key military backer of Ukraine, committing tens of billions of dollars in security assistance since the start of Russia's invasion.
But a squabbling Congress had not approved large-scale funding for Kyiv for nearly a year and a half.
Ukraine's military is facing a severe shortage of arms and recruits as Moscow exerts constant pressure from the east, with frontline circumstances expected to worsen in the coming weeks.
In contrast to some Republicans, Biden and the Democrats frame Ukraine aid as an investment in US security against future Russian aggression targeting other countries.
The Bill signed by Biden also provides for much-needed humanitarian assistance to Sudan, Haiti and Gaza, with the president calling on Israel to allow aid to quickly reach Palestinians in the war-racked coastal enclave.
"We're going to immediately secure that aid and surge it... including food, medical supplies, clean water," Biden said. "Israel must make sure all this aid reaches the Palestinians in Gaza without delay."
Another provision in the legislation gives popular social media app TikTok nine months to divest from its Chinese parent company ByteDance or be shut out of the American market.
TikTok plans to fight the measure in court, with its CEO Shou Zi Chew saying in a video message on the platform that "this is a ban. A ban on TikTok and a ban on you and your voice."