The council intends to introduce ‘shared streets' on the road, which is home to many of London's major museums. The plans would mean the road and pavement being built on the same level and separated by paving slabs with raised bars rather than a kerb.
The charity believes kerbs are safer for blind and partially sighted people and has asked the council to postpone any work until further discussions have taken place.
Bridget Warr, chief executive of Guide Dogs, said: "Our decision to pursue legal action was not taken lightly but was born of the impasse that had seemingly been reached in terms of finding a solution for all users of Exhibition Road, particularly the blind and partially sighted people we represent."
It is the second time the charity has has sought a judicial review of the council's plans. Its first application was ruled to be premature by the High Court in October.
Nicholas Paget-Brown, cabinet member for transportation, environment and leisure at the council, said safety in the new design was of "paramount importance" to the council and it wanted to work with Guide Dogs over the issue.
"We believe that the new design, which will be completed in 2012, will improve accessibility and safety for all," he said.
"At present Exhibition Road is an accident blackspot and an area where the majority of disabled people are excluded from some of the world's most important cultural institutions. We are sure that Guide Dogs would not want this exclusion to continue, neither for its membership nor for the many thousands of people with other disabilities."