Abstract-In the present paper, we will propose a new and robust method of camera self-calibration having varying intrinsic parameters from a sequence of images of an unknown 3D object. The projection of two points of the 3D scene in the image planes is used to determine the projection matrices. The present method is based on the formulation of a non linear cost function from the determination of a relationship between two points of the scene with their opposite relative to the axis of abscise and their projections in the image planes. The resolution of this function with genetic algorithm enables us to estimate the intrinsic parameters of different cameras. The important of our approach reside in the use of a single pair of images which provides fewer equations, simplifies the mathematical complexities and minimizes the execution time of the application, the use of the data of the first image only without the use of the data of the second image, the use of any camera which makes the intrinsic parameters variable not constant and the use of a 3D scene reduces the planarity constraints. The experimental results on synthetic and real data prove the performance and robustness of our approach.