Multiple natural frequencies may be encountered when analyzing the essential natural vibration of a symmetric mechanical system or sub-structure system or a system with special parameters. The transfer matrix method (TMM) is a useful tool for analyzing the natural vibration characteristics of mechanical or structural systems. It derives a nonlinear eigen-problem (NEP) in general, even a transcendental eigen-problem. This investigation addresses the NEP in TMM and proposes a novel method, called the determinant-differentiation-based method, for calculating multiple natural frequencies and determining their multiplicities. Firstly, the characteristic determinant is differentiated with respect to frequency, transforming the even multiple natural frequencies into the odd multiple zeros of the differentiation of the characteristic determinant. The odd multiple zeros of the first derivative of the characteristic determinant and the odd multiple natural frequencies can be obtained using the bisection method. Among the odd multiple zeros, the even multiple natural frequencies are picked out by the proposed judgment criteria. Then, the natural frequency multiplicities are determined by the higher-order derivatives of the characteristic determinant. Finally, several numerical simulations including the multiple natural frequencies show that the proposed method can effectively calculate the multiple natural frequencies and determine their multiplicities.