Aiming at the two-stage vibration isolation decoupling optimization problems of the power assembly of diesel generator set installed on an exported diesel locomotive, it is suggested that the stiffness of the first and secondary vibration isolators is the main factor affecting the decoupling degree of a two-stage vibration isolation system. According to the decoupling theory and principle of such systems, the stiffness of the first and secondary vibration isolators is defined. The possible value is calculated on the basis of public framework and the car body′s frequency variation range, and the possible stiffness scheme is sorted using MATLAB programming. Finally, a program is used to compare the isolation effects of the stiffness determined by the decoupling theory and the optimized ones, while taking into account the optimization objectives of the maximum comprehensive decoupling degree of 12 degrees of freedom, the maximum α decoupling degree along the crankshaft rotation direction, the minimum set vibration intensity, and the minimum dynamic supporting reaction on the installation position of the secondary vibration isolators. Results show that the comprehensive isolation performance of the vibration isolators′stiffness obtained according to the vibration isolation decoupling theory and principle is generally good, but not optimal. The optimal stiffness scheme can provide a practical reference for engineering.