"The State of Israel respects the freedom of worship at Al-Aqsa and all holy places," Yoav Gallant said in a video message, referring to the third-holiest site in Islam, located in Israeli-annexed east Jerusalem.
"The month of Ramadan may be a month of jihad, and we say to everyone not to try us -- we are ready, don't make mistakes," he said in the clip posted on his Telegram channel.
His comments came amid concerns about tensions during Ramadan at the Al-Aqsa mosque compound, a flashpoint for violence during the Muslim fasting month in past years.
The compound is also Judaism's most sacred site, known to Jews as the Temple Mount.
During Ramadan, Muslims in their tens and even hundreds of thousands pray at the compound.
On Friday a spokesman for the armed wing of the Hamas militant group called on "our people" to mobilise and head towards Al-Aqsa.
Hamas's unprecedented October 7 attack on southern Israel triggered the ongoing war in Gaza, now in its six month.
The attack resulted in the deaths of about 1,160 people in Israel, mostly civilians, according to an AFP tally of Israeli official figures.
The militants also seized about 250 hostages, dozens of whom were released during a week-long truce in November.
Israel believes that 99 hostages still in militants' hands remain alive and 31 have died.
Israel's retaliatory military campaign to destroy Hamas has killed at least 31,112 people, according to the health ministry in Hamas-run Gaza.
Mediators had been hoping to secure a new truce before Ramadan began but so far there has been no breakthrough.