The idea was examined by the South Yorkshire Integrated Transport Authority (SYITA) last week, but members quickly ruled out the proposal.
Members made their decision in response to a research report which highlighted the advantages and disadvantages of allowing bikes on trams, balancing the potential advantages with the impact on vulnerable passengers.
Mick Jameson, chairman of authority, said: "Though the ITA is keen to support cycling, it was felt that due to limited space in the low-floor areas of trams, priority should be given to those passengers with pushchairs, in wheelchairs, the elderly and infirm."
The authority had been asked to consider allowing cyclists to take their bikes on the tram for a trial period, and Sheffield cycle pressure groups said "the demand was there."
Speaking before the meeting Sheffield Pedal Ready spokesman Max McCulloch said: "Some people commute from home to work using their bikes for the whole journey, but some people only want to use a bike for part of their journey."