Although field measurements and using long hydrological datasets provide a reliable method for parameters' calibration, changes in the underlying basin surface and lack of hydrometeorological data may affect parameter accuracy in streamflow simulation. The ensemble Kalman filter (EnKF) can be used as a real-time parameter correction method to solve this problem. In this study, five representative Xin'anjiang model parameters are selected to study the effects of the initial parameter ensemble distribution and the specific function form of the parameter on the EnKF parameter estimation process for both single and multiple parameters. Results indicate: (1) the method of parameter calibration to determine the initial distribution mean can improve the assimilation efficiency; (2) there is mutual interference among the parameters during multiple parameters' estimation which invalidates some conclusions of single-parameter estimation. We applied and evaluated the EnKF method in Jinjiang River Basin, China. Compared to traditional approaches, our method showed a better performance in both basins with long hydrometeorological dataset (an increase of Kling–Gupta efficiency (KGE) from 0.810 to 0.887 and a decrease of bias from −1.08% to −0.74%); and in basins with a lack of hydrometeorological data (an increase of KGE from 0.536 to 0.849 and a decrease of bias from −15.55% to −11.42%). HIGHLIGHTS We systematically evaluated the effect of initial parameter distribution on the estimation process of different parameters in the EnKF method.; We systematically compared the application of EnKF method on single vs multiple parameters.; Application showed that the EnKF method has a superior performance in both basins with long series of hydrological data and in basins with a lack of hydrometeorological data.;