To realize the in-motion alignment of the strapdown inertial navigation system (SINS) under the geographic latitude uncertainty, we propose a latitude estimation and in-motion alignment method based on the integral dynamic window and polynomial fitting (IDW-PF) and improved Kalman filter (IKF). First, the integral dynamic window (IDW) is designed to smooth out the high-frequency line motion interference and accelerometer noise. Second, the specific force integral is performed for a cubic polynomial fitting (PF) with time as an independent variable to further suppress the line motion interference. Simultaneously, the latitude is estimated according to the geometric relationship between the angle of the gravitational acceleration vectors at different moments and the latitude. Finally, the IKF based on the multi-fading factor is designed for the in-motion alignment of SINS. A simulation experiment is conducted to verify the proposed latitude estimation and in-motion alignment method. The results indicate that the latitude can be estimated well by the method based on the IDW-PF; the mean and standard deviation of the estimated latitude can achieve −0.016° and 0.013° within 300 s. The trapezoidal maneuvering path is optimal when IKF is used, the pitch error is 0.0002°, the roll error is 0.0009° and the heading error is −0.0047° after the alignment ends at 900 s.