The deal comes after Lithuania asked fellow EU member states for assistance against illegal migration.
The Czech Republic has pledged to support the construction of a border fence between Lithuania and Belarus to combat illegal migration.
On Tuesday, the Czech government said it would contribute €530,000 to Lithuanian to help expedite the fence.
Lithuanian Interior minister Agne Bilotaite and Czech foreign minister Jakub Kulhanek signed a deal on the matter in Vilnius.
The move comes after Lithuania asked fellow EU member states and the bloc’s border agency Frontex for assistance.
Lithuania has faced an influx of thousands of mostly Iraqi migrants in the past few months streaming from neighbouring Belarus.
The EU has accused Belarusian President Alexander Lukashenko of encouraging the migrant flow in retaliation for sanctions against his country.
"Lukashenko continues to weaponize illegal migration as a means to blackmail Lithuania and by extension, the EU," Kulhanek said on Tuesday. "He has to be stopped."
Meanwhile, Bilotaite expressed gratitude to the Czech minister on Tuesday for the country's "solidarity and assistance".
In August, the European Commission pledged €12 million to Lithuania and neighboring Estonia, while Ukraine has sent hundreds of kilometers of barbed wire to the country.
Denmark has offered to sell 15 kilometres of barbed wire "to strengthen the Lithuanian border efforts along the Belarus border," Danish Immigration Minister Mattias Tesfaye told the Danish Parliament. Denmark also offered housing containers, immigration experts, and sanitation equipment to the Baltic country.
Other EU member states including Cyprus and Malta have decided to send humanitarian assistance to Lithuania.
But according to the Lithuanian Interior Ministry, the Czech Republic is the first country to offer financial assistance, which will be used for the construction of the new 508-kilometre-long border fence.
Poland has also declared a state of emergency in two regions bordering Belarus, following an increase in illegal migration
Meanwhile, the UN's migration agency says it is concerned by "dire conditions" facing migrants who have been stranded for weeks at the border between the two countries.