Queue length estimation is of great importance for measuring traffic signal performance and optimizing traffic signal timing plans. With the development of connected vehicle (CV) technology, using mobile CV data instead of fixed detector data to estimate queue length has become an important research topic. This study focuses on real-time queue length estimation for an isolated intersection with CV data. A Kalman filtering method is proposed to estimate the queue length in real time using traffic signal timing and real-time traffic flow parameters (i.e., saturated flow rate, traffic volume, and penetration rate), which are estimated using CV trajectories data. A simulation intersection was built and calibrated using field data to evaluate the performance of the proposed method and the benchmark method. Results show that when the CV penetration rate is at 30%, the average values of mean absolute errors, mean absolute percentage errors, and root mean square errors are just 1.6 vehicles, 20.9%, and 2.5 vehicles, respectively. The performance of the proposed model is also better than the benchmark method when the penetration rate of CVs is higher than 20%, which proves the validity of the proposed method. Furthermore, sensitivity analysis indicates that the proposed method requires a high penetration rate of at least 30%.