The wife of 75-year-old John Short said her husband was arrested in Pyongyang, the North Korean capital, on Sunday and had been open about his religious work on his second trip to the isolated country.
Karen Short said: "He won't be intimidated by the communists."
North Korea has held American missionary Kenneth Bae for more than a year and convicted him of trying to overthrow the state. A North Korean court sentenced Mr Bae to 15 years' hard labour and efforts by Washington to secure his release have been thwarted.
Mrs Short's wife said of her husband's arrest: "I'm not upset, we're Christian missionaries and we have tremendous support for what we do."
A UN report issued on Tuesday cited lack of religious freedom in North Korea, whose human rights abuses it likened to those of Nazi Germany.
The Australian Embassy in Seoul said it was aware of his case.