Since passive vibration isolation device cannot adaptively change the parameters of vibration isolation, as well as the high cost and practice difficulty of a large vibration isolation project, the damping parameter optimization of Magneto-rheological isolator is proposed based on the dimensionless analysis method. In order to improve the whole effect of the vibration isolation system, it must build optimization model from the angel of the dimensionless. Three target functions are built by considering the vibration transmissibility ratio, vibration displacement and vibration acceleration at the same time. According to the dynamic response characteristics of the vibration isolation system, the system multi-objective problem is solved by global optimization principle. Then the best magneto-rheological equipment damping parameters and the best drive current can be obtained. A magneto-rheological vibration isolation system under multi-frequency and multi-amplitude exciting force are simulated in MATLAB. The Root Mean Square of acceleration is decreased by 31.41% and the Root Mean Square of the force transmitted to the foundation is decreased by 58.75%. Then small scale isolation equipment based on dimensionless similarity theory is built in laboratory. The parameters optimization test results show that the vibration acceleration and the force transmitted to the foundation is significantly reduced. From the simulation and bench mutual authentication, the dimensionless parameter optimization method is effective and feasible in engineering vibration isolation.