Low-frequency vibrations of seats in vehicles, ships, aircraft, and other means of transportation severely affect the comfort and health of occupants. Additionally, the precision instruments precision instruments mounted on these platforms require effective isolation from micro-amplitude vibrations to ensure their operational accuracy. However, traditional linear vibration isolation systems have limited performance in the low-frequency range, making it difficult to meet the demands for high comfort and high-precision vibration isolation. Negative stiffness structures achieve high static stiffness and low dynamic stiffness through geometric nonlinearity, which can significantly improve low-frequency vibration isolation performance.?To address the limitation of single-stage negative stiffness systems in suppressing the secondary transmission of vibrations, a dual-stage negative stiffness vibration isolation system?is proposed. The dynamic equations of the system?are derived?based on the harmonic balance method, and the accuracy of the analytical results?is verified?through numerical methods. Parametric analysis?shows?that the system?outperforms traditional linear systems in terms of low-frequency vibration isolation performance. On this ba-sis, an evaluation index for the vibration isolation performance of precision equipment based on the peak control of acceleration transmissibility, together with a human comfort index considering the weighted mean of transmissibility, were proposed. The results?provide?a theoretical basis and a comfort e-valuation method for the design of new low-frequency vibration isolation systems for seats and precision instruments in transportation systems.