Her Majesty, who looked stylish in her pink coat and matching hat featuring a flower decoration, was seen using a walking stick for the second time this week.
The Prince of Wales and The Duchess of Cornwall watched on as the Queen delivered her speech.
She took the opportunity to praise the efforts of the key workers and volunteers who served their communities in the Covid pandemic.
She said: “I have spoken before about how recent times have, in many ways, brought us closer together.
“We all owe a debt of gratitude to those who have risen so magnificently to the challenges of the last 18 months – from key workers to volunteers, who have done so much to serve their communities.
“They are shining examples of the spirit for which the Welsh people are so renowned, a spirit which I have personally encountered so many times.”
She took a lift to a lobby area of the parliament building in Cardiff where she met dignitaries before attending the opening.
Mark Drakeford, First Minister of Wales, said the sixth session of the Senedd was a period for the people of Wales to “look to the future”.
He said he wanted to meet the “challenges” ahead by promoting “prosperity, equality and well-being for everyone in Wales”.
In his speech, he said: “We have seen the best of Wales in our health and care system, the shop workers, our teachers, all those public service workers, the businesses and volunteers, whose commitment, dedication and sheer hard work has helped to keep Wales open during this most difficult of times.”
He added: “As we look beyond the pandemic, we recognise that we still have many challenges ahead.
“To meet them we will use all of our powers to promote prosperity, equality and well-being for everyone in Wales. And to turn ourselves to that other great crisis of our times, a crisis in climate change and the loss of biodiversity.”