In this paper, a new approach based on the continuum model is proposed to estimate the main cable tension force of suspension bridges from measured natural frequencies. This approach considered the vertical vibration of a main cable hinged at both towers and supported by an elastic girder and hangers along its entire length. The equation reflected the relationship between vibration frequency and horizontal tension force of a main cable was derived. To avoid to generate the additional cable tension force by sag-extensibility, the analytical solution of characteristic equation for anti-symmetrical vibration mode of the main cable was calculated. Then, the estimation of main cable tension force was carried out by anti-symmetric characteristic frequency vector. The errors of estimation due to characteristic frequency deviations were investigated through numerical analysis of the main cable of Taizhou Bridge. A field experiment was conducted to verify the proposed approach. Through measuring and analyzing the responses of a main cable of Taizhou Bridge under ambient excitation, the horizontal tension force of the main cable was identified from the first three odd frequencies. It is shown that the estimated results agree well with the designed values. The proposed approach can be used to conduct the long-term health monitoring of suspension bridges.