Biden signed the legislation while in South Korea.
The Senate voted overwhelmingly in favor of the package, 86-11, with all but 11 Republicans siding with Democrats in the vote. The bill also had broad bipartisan support in the House in its 368-57 vote earlier this month.
Biden asked for $33 billion in new aid for Ukraine earlier this month and the House increased the proposal to a $40 million package after adding more defensive and humanitarian funding.
The United States has given billions of dollars in aid to Ukraine since the Russian invasion began on Feb. 24. That aid, however, has just about run out.
About a fifth of the money in the bill would to go restocking U.S. military equipment sent to Ukraine. Some senators raised concerns about giving Ukraine — a former Soviet republic located right next to Russia — more weapons like stinger and Javelin missiles.
The package includes $6 billion in assistance for training, equipment, weapons and logistics support as well as $8.7 billion to replenish U.S. stocks of military equipment that the Biden administration has already sent.
More than $5 billion is included to alleviate global food insecurity, $3.9 billion for European Command operations, $900 million allotted to refugee support services for arrivals from Ukraine and nearly $14 billion for the State Department to aid Kyiv.
Also this week, Biden authorized an additional $100 million in military assistance for Ukraine, the 10th drawdown of equipment for Kyiv since the start of the war.
The Defense Department said in a statement that the equipment going to Ukraine includes 18 155mm howitzers, 18 tactical vehicles to tow the howitzers, three counter-artillery radars and field equipment as well as spare parts.