In order to realize more efficient and accurate automatic focusing of optical fiber end face, the definition evaluation function of existing images is studied. On the basis of this, a new definition evaluation function with simple calculation and good anti-noise is proposed. An optical fiber geometry measurement system is used to obtain 24 frames of equally spaced optical fiber end-face image. Different image definition evaluation functions are used to calculate the 24 frames of images. The corresponding focusing curves are obtained and their monomorphism, monotonicity and anti-noise are analyzed. The results show that the proposed evaluation function has better performance and has certain reference value for automatic focusing technology.