Since before the American Revolution Fishkill's Trinity Church had been the only Episcopal house of worship in southeastern Dutchess County. It was not easily accessible to residents of the hamlets of Matteawan and Fishkill Landing, today's Beacon. So, in 1832, three local sisters began offering religious instruction to children at their house, today's Madam Brett Homestead. Their parents and other adults met in a dry goods store, and by the end of the year the group had reached the size where it could form a parish of its own. In 1833 it formally incorporated as St. Anna's, and the next year built a small brick church with columned portico, at what is now the intersection of Main Street and Tioronda Avenue, on donated land.

In the following decades, Tioronda became the railroad's right-of-way, making it a less desirable location so the congregation decided in 1863 to build a new church elsewhere. One congregant, Charles Wolcott, gave six acres next to the church's then-cemetery. In 1866 he added the adjacent land of equal size then used as a cemetery by a local Presbyterian church.

The deed stipulated that a religious edifice of at least $20,000 in value ($259,000 inflation adjusted 2008 value) had to be built on the property within three years of its conveyance to the church. To raise the money, the church sold both its old church and rectory.

Mr. Frederick Clarke Withers, who had already designed the Dutch Reformed Church of Fishkill Landing and the Eustatia home of John Monell, one of St. Anna's vestrymen, was easily recruited to design the new church and rectory due to his involvement and relationships with members of the church. Mr. Henry Winthrop Sargent was also involved in raising funds for the new building and at the time of St. Luke's construction was also a member of the vestry.

Ground was broken in 1868, and the church officially became St. Luke's Church of Matteawan and the first service was held the following year. The other buildings included the rectory and a small frame schoolhouse where the Parish House now stands. The total construction cost came to $60,000 ($770,000 in 2008 dollars). This debt took a while to retire, and as a result the church was not formally consecrated until 1879.

In 1887, fire severely damaged the rectory. The outside was salvaged, but the interior had to be rebuilt. The original floor plan was recreated, but some more contemporary decor was added. Five years later, in 1892, the church decided to demolish the original schoolhouse and replace it with the Parish House. The building was supposed to be cross-shaped, but that design could not be completed as there was not enough money to complete its construction. The addition of the Great Hall in the 1960's finally completed the original design. During that period, the church building itself also got a new heating system, carpet for the floor under the pews, and the current roof tile.