The Lothians MSP was the sole candidate to replace Ken Macintosh in the role, and was elected by 97 MSPs, with 28 voting against, two abstentions and one spoiled ballot paper.
Ms Johnstone said she was very proud to be Presiding Officer and it was a "major challenge at an incredibly important time.”
She added: "I joined Scotland Forward, the campaign to have a devolved parliament before I joined the Green party, and this is a very important place for me. I was fortunate to get a job with Robin Harper, the first Green parliamentarian, so I’ve been in the parliament in a number of guises and it’s been a privilege to serve as a Scottish Green representative.
"It is the duty of government and parliament to do all we can to ensure those living in Scotland have the best lives they possibly can. I want to encourage a culture of open debate but in an atmosphere of inclusivity, tolerance and respect.”
She welcomed the diversity of the new MSPs as “fabulous progress” but said “we can do better, we need to get to a stage where this parliament truly represents all people in Scotland, so we really mirror the people in our neighbourhoods.
“We are also in the midst of a climate emergency – our young people have done such a fabulous job bringing this issue to life, they have asked us to debate it on their behalf and to act. We only have a few years to act and its important Scotland embraces the opportunity COP26 will provide.”
After her thank you speech, the first action of Ms Johnstone was to suspend the meeting of Parliament until 10.30am tomorrow when the election of two deputy presiding officers will begin.
Scottish Greens co-leaders Patrick Harvie and Lorna Slater said: “We congratulate Alison Johnstone on her election, and we are confident that she will be an even-handed moderator as Parliament addresses the pressing challenges that Scotland faces over the next five years.”