The move to ban the smoking devices came following health advice.
Sales of e-cigarettes, battery-powered sticks that release nicotine when inhaled, have soared in recent years. New figures released this week show that the number of adult smokers in Scotland using e-cigarettes has risen from 3% in 2010 to 17% in 2014.
Experts say it is not yet known what harm the tobacco-free devices could inflict.
The decision to ban their use at Holyrood was made by the Scottish Parliamentary corporate body at a meeting last week after guidance from health officials.
It has been welcomed by campaigners who have called for the market in e-cigarettes to be regulated. Among the measures they want are a restriction on sales to children and their promotion.
A spokesman for the Royal Pharmaceutical Society in Scotland said: "We commend the Parliament applying the same restrictions to e-cigarettes as smoking in the Scottish Parliament."
Firms including ScotRail, Starbucks and Wetherspoon pubs have already banned customers using e-cigarettes, and their use has been prohibited in and around Commonwealth Games venues in Glasgow.
The British Medical Association has called for a ban on e-cigarettes in all enclosed public places.
A Scottish Parliament spokesman said: "The corporate body's decision to treat e-cigarettes in this way is based on a precautionary approach in line with advice issued to all Scottish NHS health boards by the Scottish directors of public health."