In the multifocus microscopic image measurement method, the distortion of the three-dimensional (3D) reconstruction model has always been an important factor affecting the measurement result. In spatial domains, the focus measure algorithm is based on the gradient change of the pixel point to determine the degree of focus of the pixel. So it will be difficult to accurately extract the focus of the pixel in the areas where color difference is not obvious, resulting in 3D model distortion. According to the optical principle, the high-frequency coefficients of the clear image are larger than the high-frequency coefficients of the blurred image. Based on this characteristic, this paper proposes a new multifocus microscopic image 3D reconstruction algorithm using a nonsubsampled wavelet transform (NSWT). The NSWT does not consider the downsampling in wavelet decomposition and has translational invariance. Therefore, the wavelet transform value of each pixel can be calculated in the image, so the high-frequency coefficient of each pixel can be obtained; then the convolution calculation is performed on the high-frequency coefficients of the pixel points in the fixed window as the focus measure value of the pixel point. Compared with the traditional algorithm, the algorithm proposed in this paper can show better unimodal and antinoise performance on the focusing measure curve. In this paper, the reconstruction of the experimental object is Alicona standard block triangular and semicylindrical. The proposed algorithm and the traditional algorithm for comprehensive measure use the root mean square error, peak signal to noise ratio, and correlation coefficient as the measure index. The experimental results and comparative analysis prove the correctness of the proposed algorithm and enable more accurate reconstruction of 3D models based on multifocus microscopic images.