The focusing evaluation method based on wavelet transform and adaptive genetic algorithm (AGA) is developed in this paper. First, db7 wavelet transform is performed and the corresponding images are evaluated. Afterward, the optimal evaluation region is searched automatically in 2D wavelet coefficients by AGA. Finally, the criteria function based on the weighted MWT2 operator is used to evaluate the focusing condition. The wavelet base, order number of vanishing moments, and wavelet decomposition level are the key factors in constructing the operator. The parameters of new focus measure operator are analyzed and verified through experimental methods. The comparative experiments with other methods show that the AGA method not only could find the approximate optimal evaluation window quickly and accurately, but it has better generality as well. The weighted evaluation function of the MWT2 operator based on the db7 wavelet has higher sensitivity and better real-time performance.