The invention discloses a motorcycle frame actual running load spectrum fatigue reproduction test method. The steps include: (1) collecting load spectrum of a motorcycle frame in actual running; (2) mounting the motorcycle frame in a test system; (3) calculating frequency response functions between each response point and each exciting point; (4) selecting a response function between expected response signals and an analog iteration frequency; (5) calculating initial driving signals xd(t) of each vibration exciter; (6) enabling xd(t) as driving signals of the vibration exciters, collecting response signals of expected response points, comparing the response signals with the expected response signals, revising the response signals to enable the response signals to be the same as the expected response signals, and recording the final signals xd(t); and (7) adopting the final xd(t) as an input to perform fatigue tests on the motorcycle frame. The method combines actual running stress condition of the motorcycle frame, performs motion analysis and decoupling analysis, selects multi-point loading and force control modes, and accurately and efficiently reproduces load spectrum and fatigue situations during actual running of the motorcycle frame.