As a sensing device, microfiber knot resonator (MKR) has been widely used in sensing field due to its advantages of high sensitivity and fast response. It is important to analyze the spectrum of MKR for detecting environmental parameters in that the spectrum can reflect the change of ambient environment. In this paper, the relationship between spectrum of MKR and seawater temperature has been studied theoretically and experimentally. Firstly, the dependences of the propagation constants of fundamental mode (HE11) on the fiber diameter and the probing wavelength are studied theoretically in this paper, including two orthogonal polarization states of fundamental mode. The calculated results show that the propagation constant increases with the increasing fiber diameter and the decreasing probing wavelength. Simultaneously, the dependence of propagation constant of fundamental mode on seawater temperature is studied. The results show that the larger propagation constant corresponds to the higher seawater temperature, which indicates that the seawater temperature affect the mode propagation constant. Thus, the seawater temperature can be obtained by detecting the resonant spectrum. Secondly, the experimental system for seawater temperature sensing is set up, with which the resonant spectrum of MKR for seawater temperature sensing are obtained. The experimental results show that two sets of resonant peak exist on the spectrum, which are corresponding to TE and TM modes of fundamental mode. The sensing sensitivities of the two polarization modes are 5.54 pm/°C and 5.24pm/°C, respectively. Finally, the reason for resonant spectrum of the two polarization states is discussed, which is that the separation of the two modes resulting from the twisted coupler of the knot zone, and the resonant intensity of the two polarization modes is analyzed. The resonant intensities of the two modes are different and one increases while the other one decreases with the increasing probing wavelength. It is determined by the coupling coefficients and attenuations of the two coupled states, which are affected by the probing wavelength. The experimental and theoretical results agree well.