To improve the estimation accuracy of observation pulse phase， the relationship between pulsar observation period and observation pulse phase estimation accuracy in navigation is studied. Firstly， the theoretical lower bound of observation pulse phase estimation variance at spacecraft is derived， and then the relationship between observation pulse phase estimation error and pulsar observation period is given. Taking the minimum mean square error of observation pulse phase estimation as the criterion， the approximate calculation formula of the optimal observation period is given. Finally， the observational data of PSR B0531+21 are used to verify the correctness of the formula. The simulation results show that the prediction error of the given optimal observation period formula for PSR B0531+21 is 44 s， which proves the correctness of the formula given in this paper and provides a theoretical basis for determining the observation period of pulsars in navigation.