Figures show staff receiving such packages rose from 326 in 2013-14 to 413 in 2014-15.
Transport union TSSA said London Mayor Boris Johnson was creating a "first class gravy train" for senior managers.
But TfL said remuneration consultants had found its pay was "significantly below the market level".
It said in many cases it was severance payments and overtime, which pushed the amount into six figures.
It said: "The overtime was worked by specialist engineers and highly skilled project staff, 91 of whom earn a base salary of less than £100,000.
"However, the overtime earned took their total remuneration above this."
TSSA leader Manuel Cortes said: "While those earning over £100,000 a year goes through the 400 barrier for the first time, our members at London Underground have been offered less than RPI (inflation) - 0.75% for this year."
TfL also announced the base pay of London's Transport Commissioner Sir Peter Hendy, who was on around £348,000 in 2010, had been frozen for a sixth year in a row.
London Assembly Green Party member Darren Johnson said he welcomed the cap on Sir Peter's pay but added that TfL "needs to do more to hold the line on pay costs at the top end of their organisation".
The number of staff working for Transport for London (TfL) and earning more than £100,000 has reached more than 400 for the first time.