It is the first so-called red line in any post-election negotiations to have been set out by Plaid.
First Minister Carwyn Jones has said proposals for the relief road would be included in Labour's manifesto.
The new Â£1bn six-lane stretch of motorway - the so-called black route - would run south of Newport.
It would be the biggest infrastructure project carried out by the Welsh government since the start of devolution.
But others, including Plaid, have suggested upgrading an existing stretch of road should be carried out instead at a much lower cost.
On Wednesday the Welsh Conservatives said they would have "diggers in the ground" to build a planned road within 12 months, although they have yet to decide on a specific route.
Ms Wood told BBC Wales' Sunday Politics Wales programme: "I have been very clear since I've been leader of this party that we have to spread wealth and prosperity throughout the nation, and focusing spending on one small corner of the nation is completely unacceptable to Plaid Cymru.
"So we would not support any government that wanted to go ahead with that project."
Plaid Cymru leader Leanne Wood has ruled out any kind of deal with Labour after the election if the party pushes ahead with plans for an M4 relief road.